FINAL Report on Carcinogens Background Document...

FINAL

Report on Carcinogens Background Document for

Styrene

September 29, 2008

U.S. Department of Health and Human Services Public Health Servces

National Toxicology Program Research Triangle Park, NC 27709

This Page Intentionally Left Blank

RoC Background Document for Styrene

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FOREWORD

The Report on Carcinogens (RoC) is prepared in response to Section 301 of the Public

Health Service Act as amended. The RoC contains a list of identified substances (i) that

either are known to be human carcinogens or are reasonably be anticipated to be human

carcinogens and (ii) to which a significant number of persons residing in the United

States are exposed. The Secretary, Department of Health and Human Services (HHS), has

delegated responsibility for preparation of the RoC to the National Toxicology Program

(NTP), which prepares the report with assistance from other Federal health and

regulatory agencies and nongovernmental institutions.

Nominations for (1) listing a new substance, (2) reclassifying the listing status for a

substance already listed, or (3) removing a substance already listed in the RoC are

reviewed in a multi-step, scientific review process with multiple opportunities for public

comment. The scientific peer-review groups evaluate and make independent

recommendations for each nomination according to specific RoC listing criteria. This

background document was prepared to assist in the review of styrene. The scientific

information used to prepare Sections 3 through 5 of this document must come from

publicly available, peer-reviewed sources. Information in Sections 1 and 2, including

chemical and physical properties, analytical methods, production, use, and occurrence

may come from published and/or unpublished sources. For each study cited in the

background document from the peer-reviewed literature, information on funding sources

(if available) and the authors’ affiliations are provided in the reference section. The draft

background document was peer reviewed in a public forum by an ad hoc expert panel of

scientists from the public and private sectors with relevant expertise and knowledge

selected by the NTP in accordance with the Federal Advisory Committee Act and HHS

guidelines and regulations. This document has been finalized based on the peer-review

recommendations of the expert panel and public comments received on the draft

document. Any interpretive conclusions, comments, or statistical calculations made by

the authors or peer reviewers of this document that are not contained in the original

citation are identified in brackets [ ].


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A detailed description of the RoC nomination review process and a list of all substances

under consideration for listing in or delisting from the RoC can be obtained by accessing

the 12th RoC at http://ntp.niehs.nih.gov/go/9732. The most recent RoC, the 11th Edition

(2004), is available at http://ntp.niehs.nih.gov/go/19914.


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CONTRIBUTORS

Project Managers, Authors, and Principal Reviewers

National Toxicology Program (NTP) and National Institute of Environmental Health Sciences (NIEHS)

Ruth Lunn, Dr.P.H. Director, Report on Carcinogens Group C.W. Jameson, Ph.D. Report on Carcinogens Office (former

Director; currently at CWJ Consulting, LLC)

Gloria Jahnke, D.V.M. Health Scientist, Report on Carcinogens Group

Constella Group, LLC (Support provided through NIEHS Contract Number NO1-ES-35505) Sanford Garner, Ph.D. Principal Investigator Stanley Atwood, M.S., DABT Greg Carter, M.E.M. Andrew Ewens, Ph.D. Dana Greenwood, B.S. Jennifer Ratcliffe, Ph.D.

Consultants Henrik Kolstad, M.D., Ph.D. Arbejdsmedicinsk Klinic, Department of

Occupational Medicine, Aarhus, Denmark

Pavel Vodicka, M.D., Ph.D. Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Czech Republic

Joe Haseman, Ph.D. Independent Consultant Doug Rickert, Ph.D. Independent Consultant

Administrative Support Ella Darden, B.S. Constella Group, LLC Tracy Saunders, B.S. Constella Group, LLC Shawn Jeter, B.S. Report on Carcinogens Group, NIEHS Jenaya Brown Report on Carcinogens Group, NIEHS

Editorial Support Susan Dakin, Ph.D. Independent Consultant in Technical &

Scientific Writing & Editing


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PEER REVIEW

The draft background document on Styrene was peer reviewed by the Report on Carcinogens (RoC) expert panel for Styrene. The panel met in a public forum at the Radisson Hotel, Research Triangle Park, NC on July 21-22, 2008. Members of the expert panel are as follows:

David Phillips, Ph.D., DSc. FRCPath (Chair) Institute of Cancer Research

Scot Eustis, D.V.M., Ph.D., DACVP Independent Consultant

Peter Infante, Dr.P.H., M.PH., D.D.S. Peter Infante Consulting, Inc.

Genevieve Matanoski, M.D., Dr.P.H. Johns Hopkins Bloomberg School of Public Health Department of Epidemiology

Shane S. Que Hee, Ph.D. University of California, Los Angeles School of Public Health, Department of Environmental Health Sciences

Thomas J. Smith, Ph.D., CIH Harvard School of Public Health Department of Environmental Health

Suzanne Snedeker, Ph.D. Cornell University College of Veterinary Medicine

Michael P. Stone, Ph.D. Vanderbilt University Department of Chemistry

Elizabeth M. Ward, Ph.D. American Cancer Society Epidemiology and Surveillance Research

Garold S. Yost, Ph.D. University of Utah Department of Pharmacology and Toxicology

Lauren Zeise, Ph.D. California EPA OEHHA Reproductive and Cancer Hazard Assessment


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Criteria for Listing Agents, Substances or Mixtures in the Report on Carcinogens U.S. Department of Health and Human Services

National Toxicology Program The criteria for listing an agent, substance, mixture, or exposure circumstance in the RoC are as follows:

Known To Be Human Carcinogen:

There is sufficient evidence of carcinogenicity from studies in humans*, which indicates a causal relationship between exposure to the agent, substance, or mixture, and human cancer.

Reasonably Anticipated To Be Human Carcinogen: There is limited evidence of carcinogenicity from studies in humans*, which indicates that causal interpretation is credible, but that alternative explanations, such as chance, bias, or confounding factors, could not adequately be excluded, or there is sufficient evidence of carcinogenicity from studies in experimental animals, which indicates there is an increased incidence of malignant and/or a combination of malignant and benign tumors (1) in multiple species or at multiple tissue sites, or (2) by multiple routes of exposure, or (3) to an unusual degree with regard to incidence, site, or type of tumor, or age at onset, or there is less than sufficient evidence of carcinogenicity in humans or laboratory animals; however, the agent, substance, or mixture belongs to a well-defined, structurally related class of substances whose members are listed in a previous Report on Carcinogens as either known to be a human carcinogen or reasonably anticipated to be a human carcinogen, or there is convincing relevant information that the agent acts through mechanisms indicating it would likely cause cancer in humans.

Conclusions regarding carcinogenicity in humans or experimental animals are based on scientific judgment, with consideration given to all relevant information. Relevant information includes, but is not limited to, dose response, route of exposure, chemical structure, metabolism, pharmacokinetics, sensitive sub-populations, genetic effects, or other data relating to mechanism of action or factors that may be unique to a given substance. For example, there may be substances for which there is evidence of carcinogenicity in laboratory animals, but there are compelling data indicating that the agent acts through mechanisms which do not operate in humans and would therefore not reasonably be anticipated to cause cancer in humans.

*This evidence can include traditional cancer epidemiology studies, data from clinical studies, and/or data derived from the study of tissues or cells from humans exposed to the substance in question that can be useful for evaluating whether a relevant cancer mechanism is operating in people.



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Executive Summary Introduction 1

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Styrene is a viscous, highly flammable liquid used worldwide in the production of

polymers, which are incorporated into products such as rubber, plastic, insulation,

fiberglass, pipes, automobile parts, food containers, and carpet backing.

Styrene was nominated for possible listing in the Report on Carcinogens by a private

individual based on its widespread use and exposure and evidence of carcinogenicity

from studies in humans and experimental animals.

Human Exposure

The primary use of styrene is in the manufacture of polystyrene, which is used

extensively in the manufacture of plastic packaging, thermal insulation in building

construction and refrigeration equipment, and disposable cups and containers. Styrene

also is used in styrene-butadiene rubber, other polymers, and resins that are used to

manufacture boats, shower stalls, tires, automotive parts, and many other products. U.S.

production of styrene has risen steadily over the past 70 years, with 11.4 billion pounds

produced in 2006 (domestic production capacity for 2006 was estimated at 13.7 billion

pounds). Styrene and styrene metabolites in blood and urine, and styrene-7,8-oxide–DNA

adducts and styrene-7,8-oxide–hemoglobin adducts are generally accepted biological

indices of exposure to styrene. The primary source of exposure to the general public is

inhalation of indoor air; however, exposure can also occur from inhalation of outdoor air,

ingestion of food and water, and potentially from skin contact. Tobacco smoke also can

be a major source of styrene exposure for both active smokers and individuals exposed to

environmental tobacco smoke. Outdoor and indoor air levels (including air levels in most

other occupational settings) are generally below 1 ppb [0.001 ppm], although higher

levels have been reported. Workers in certain occupations, including the reinforced-

plastics , styrene-butadiene, and styrene monomer and polymer industries, are potentially

exposed to higher levels of styrene than the general public. Air levels in the reinforced-

plastics industry are generally lower than 100 ppm, [although much higher levels have

frequently been measured], while levels in the styrene-butadiene industry and the styrene


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monomer and polymer industries have rarely been reported to exceed 20 ppm. Numerous

Federal agencies have established regulations for styrene, including the Department of

Homeland Security, DOT, EPA, FDA, and OSHA, and both ACGIH and NIOSH have

established guidelines to limit occupational exposure to styrene.

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Human Cancer Studies

Numerous epidemiological studies have evaluated the relationship between styrene and

cancer in humans. Most of the studies are cohort studies of workers in three major

industries: (1) the reinforced-plastics industry, (2) the styrene-butadiene rubber industry,

and (3) the styrene monomer and polymer industry. Two additional cohort studies (one

on biomonitored workers, and the second on environmental exposure to styrene-

butadiene), several case-control studies, and an ecological study have also been

published.

The limitations of these studies include potential misclassification of styrene exposure

and disease, small numbers of long-term workers, inadequate follow-up, and the potential

for co-exposure to other chemicals. Thus, although more than a hundred thousand

workers have been studied to assess a possible carcinogenic effect of styrene exposure,

only a small fraction of well-characterized, high-level, and long-term styrene-exposed

workers have been followed for a sufficiently long time. In addition, most of the available

studies of occupational cohorts have focused only on male workers (who constitute the

majority of exposed workers) or have not performed gender-specific risk analyses. [Thus,

comparatively few data are available on cancer incidence or mortality among exposed

female workers, limiting the ability to evaluate breast cancer or cancers at tissue sites

specific for females.]

Workers in the reinforced-plastics industry have the highest levels of exposure and few

other potentially carcinogenic exposures, but many of the workers in this industry have

short-term exposure, often of less than a year. Cancer mortality or incidence was studied

in the following four populations of reinforced-plastics workers: (1) in Washington state

in the United States (Ruder et al. 2004), (2) in 30 manufacturing plants in unspecified

U.S. locations (Wong et al. 1994), (3) in Denmark (Kolstad et al. 1994), and (4) in


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Europe (Denmark, Finland, Italy, Norway, United Kingdom, and Sweden) (Kogevinas et

al. 1994a). (The Danish and the European populations were partly overlapping, as 13,682

Danish male workers were included among the 36,610 male workers making up the

European cohort.)

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In the styrene-butadiene industry, the cohort studies are among the largest, with the

longest follow-up times. The principal methodological challenge is to separate the

potentially independent or synergistic effects of butadiene, a known human carcinogen,

which is highly correlated with styrene in this industry. Two independent (non-

overlapping populations) are available, a small cohort of 6,678 male workers at a rubber

tire manufacturing plant (a subset of the workers were engaged in the production of

styrene-butadiene and other rubbers) (McMichael et al. 1976a) and a larger cohort

established by Delzell and colleagues (Delzell et al. 1996, 2006) of 13,130 to 16,610

styrene-butadiene rubber industry workers from multiple plants in the United States and

Canada. The cohort established by Delzell includes most (but not all) of the workers from

two cohorts ― a 2-plant cohort (Texas) (Meinhardt et al. 1982) and an 8-plant cohort

originally established by Matanoski and colleagues (United States and Canada) and

reported in a series of previous publications (7 of the 8 plants were included in the

Delzell cohort). Thus, there is considerable overlap between these populations. Two

nested case-control studies (Matanoski et al.1997, Santos-Burgoa et al. 1992) of a single

group of cases with lymphohematopoietic cancers were available from the Matanoski

cohort. The Delzell cohort expanded the previous cohorts to include workers employed

from 1943 to January 1, 1991 and followed to 1998, whereas the earlier cohort included

workers employed until 1976 and followed until 1982. In addition, the individual study

populations were established by different procedures and exclusion criteria (which may

partly explain the lack of complete consistency in the number of study subjects across the

published studies) and often used different exposure assessments, selection of study

subjects, and types of analysis. Two types of analyses were conducted on the Delzell

cohort: external analyses reporting on standardized mortality ratios (SMRs) for the total

cohort or subsets of the cohorts for multiple cancers sites (Sathiakumar et al. 1998,

2005), and, secondly, internal analyses of relative risk (RR) estimates for quantitative

exposure to styrene and lymphohematopoietic cancers (Delzell et al. 2001, 2006,


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Macaluso et al. 2006, Graff et al. 2005). (Dimethyldithiocarbamate [DMDTC] was also

included as a potential confounder in some analyses of lymphohematopoietic cancer in

the Delzell cohort, according to the authors, because of its potential immunosuppressant

activity in CD4+ lymphocytes, although its carcinogenicity has not been evaluated

outside of this series of studies). Workers in the styrene monomer and polymer industry

may be exposed to a variety of chemicals, including benzene, toluene, ethylbenzene, and

various solvents, and the cohorts are smaller, with many short-term workers, and few

cancer outcomes.

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The potential effect of styrene on lymphohematopoietic cancers has been studied most

extensively. Findings for lymphohematopoietic cancer and other tumor sites of interest

are discussed below.

Lymphohematopoietic cancers

Statistically significant increases were observed for all lymphohematopoietic cancers

combined and leukemia among rubber-tire manufacturing workers (McMichael et al.

1976) and statistically nonsignificant increases were observed for combined

lymphohematopoietic cancers and some specific lymphohematopoietic cancers in the

Meinhardt and Matanoski cohorts, but the potentially confounding effects of butadiene

and other exposures were not analyzed. Two nested case-control studies (using different

types of analyses and exposure assessments and the same group of cases) from the

Matanoski cohort attempted to evaluate the relative contribution of styrene and butadiene

to lymphohematopoietic cancer mortality. Santos-Burgoa et al. (1992) found no

significant excess risks for combined and specific lymphohematopoietic cancers and

mean exposure after controlling for butadiene exposure. Matanoski et al. (1997)

calculated risks for both average and cumulative exposure to styrene. Taking into account

butadiene exposure, and demographic and employment variables in step-down regression

analyses, these models found, for an average exposure of 1 ppm vs. no exposure,

significant associations for all lymphohematopoietic cancers combined, lymphomas, and

myeloma, but not leukemia. For cumulative exposure, significant positive associations

between styrene exposure and combined lymphohematopoietic cancers, leukemia, and


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myeloma were found, with butadiene exposure dropping out of each of the final models

except for leukemia.

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Specific lymphohematopoietic cancers have been studied more extensively in the Delzell

cohort. With respect to leukemia, statistically significant increases have been reported

among subgroups of workers with longer durations of employment and longer latency,

with the highest cumulative exposure, and in certain specific job groups (Sathiakumar et

al. 2005, Delzell et al. 2006). Internal analyses by Delzell et al. involving single-

chemical (styrene only), 2-chemical (styrene and butadiene), and 3-chemical (styrene,

butadiene, and DMDTC) models of cumulative exposure have shown increased relative

risks of leukemia with increasing cumulative styrene exposure. However, the response

was attenuated when controlling for exposure to butadiene and was no longer apparent

(RRs were less than or equal to one) after additionally controlling for DMDTC. Elevated

risks for leukemia were also observed with increasing exposure to styrene peaks in

single-chemical, 2-chemical and 3-chemical models (although it was attenuated

somewhat in the 2- and 3-chemical models) (Graff et al. 2005, Delzell et al. 2006).

No statistically significant increased risks were found for other lymphohematopoietic

cancers in all employees of the Delzell cohort, but statistically significant risks of NHL

and CLL combined were found among workers with higher exposure in an external

(SMR) analysis, and in internal analyses among ever-hourly workers, ever-hourly

workers with 10+ years of employment and 20 to 29 years or 30 years since first hire, and

among specific job groups. Risks of NHL or NHL and CLL combined appeared to

increase with increasing cumulative styrene exposure; the risks increased when butadiene

was added to the model, and were somewhat attenuated in models that included DMDTC.

Exposure to butadiene did not appear to be related to NHL and CLL combined or NHL

risk. [However, it should be noted that no trend analyses were performed on these data.]

(Graff et al. 2005, Delzell et al. 2006). No associations were found for other types of

lymphohematopoietic cancers and styrene exposure in the Delzell cohort.

In the reinforced-plastics industry, among the highest-exposure groups, the total number

of observed versus expected deaths or cases across the four cohorts were comparable for


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all lymphohematopoietic (52 observed vs. 52.8 expected), lymphomas (14 vs. 15.1), or

leukemia (19 vs. 19.8), and were slightly higher than expected for Hodgkin’s disease (11

observed vs. 7.9 expected) and multiple myeloma (4 vs. 3.4). Significantly increased

risks for leukemia incidence were reported in the Danish study among workers with

earlier first date of exposure, and who had worked at least 10 years since first

employment, but not for workers employed for 1 year or more (Kolstad et al. 1994). In

the European multi-country cohort (which overlaps with the Danish study), no excess of

leukemia mortality was found, and no exposure-response relationships with cumulative

or average exposure were observed, although a non-significant trend was observed with

time since first exposure (Kogevinas et al. 1994a). With respect to other

lymphohematopoietic cancers, non-significantly increased risks for non-Hodgkin’s

lymphoma were found in the Danish and European multi-country cohorts. Positive

exposure-response relationships with average styrene exposure and time since first

exposure was observed for lymphohematopoietic cancers (P = 0.019 and 0.012,

respectively) and for malignant lymphoma (P = 0.052 and 0. 072, respectively) in the

European multi-country cohort, but no relationship with cumulative exposure was

observed (Kogevinas et al. 1994a). No excesses in mortality from any

lymphohematopoietic cancers were observed in the two smaller cohort studies (Ruder et

al. 2004 and Wong et al. 1994). In the styrene monomer and polymer industries, the risk

of lymphohematopoietic malignancies was also increased in most of the studies (as well

as the total number of observed cases across studies), but these workers might also have

been exposed to benzene.

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Pancreatic cancer

Among the highest styrene-exposed group in the reinforced-plastics industry, there was

an excess in the total number of observed cases of pancreatic cancer across the four

cohort studies compared with the total number of expected cases [corresponding to an

SMR of 1.77 (95 % CI = 1.23 to 247)]. Increases in pancreatic cancer risk were observed

in three of the four reinforced-plastics industry cohorts (one of which was statistically

significant [Kolstad et al. 1995], and the other two of which were nonsignificant

[Kogevinas et al. 1994a, Ruder et al. 2004]). The risk of pancreatic cancer was slightly


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higher among the Danish workers with longer term employment and earlier start date,

and increased with cumulative exposure in the multi-plant cohort. No indications of

exposure-response relationships were found in the smaller U.S. cohorts. Statistically

nonsignificant increased risks were also observed in one study in the styrene monomer

and polymer industry (Frentzel-Beyme et al. 1978), and among biomonitored workers

(10 years after the first measurement) (Anttila et al. 1998). However, no increased risk of

pancreatic cancer was reported among styrene-butadiene workers (Sathiakumar et al.

2005).

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Esophageal cancer

Among workers with high potential exposure to styrene, increases in esophageal cancer

risk were reported in three of the four cohorts (statistically significant increases in

mortality were observed among all exposed workers in the two U.S. studies of

reinforced-plastics workers [Ruder et al. 2004, and Wong et al. 1994] and a statistically

nonsignificant increase among a subset of laminators in the European cohort [Kogevinas

et al. 1994a]). Risks were not elevated among the Danish reinforced-plastics workers

(Kolstad et al. 1994). Across the industry, an approximately 2-fold excess of esophageal

cancer was observed among high-exposed groups (laminators and others). A

nonsignificant trend with cumulative exposure was reported in the European multi-

country study. No increases in risk were reported among styrene-butadiene rubber

workers or among styrene monomer and polymer workers.

Other sites

Findings were less consistent for cancer at other sites. Significantly increased risks were

observed for cancers of the lung, larynx, stomach, benign neoplasms, cervix and other

female tumors, prostate, rectum, and urinary system in either a single study or two

studies. There were some supporting exposure-response data for cancers of the urinary

system and rectum. A significant increase in breast cancer mortality was observed in a

case-control study of occupational exposures among adult females (Cantor et al. 1995),

although there was no evidence of increased risk between low- and high-exposure

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breast cancer in the general population, but exposure estimates were based on

environmental releases of styrene, which are the least precise measures of exposure.

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Studies in Experimental Animals

The carcinogenicity of styrene in rats and mice has been investigated by several routes of

exposure. Other relevant studies in experimental animals include studies of mixtures (β-

nitrostyrene and styrene) and studies of the major metabolite of styrene, styrene-7,8-

oxide (styrene oxide).

Mice

Three strains of mice were exposed to styrene by gavage. In male B6C3F1 mice, exposure

to styrene for 5 days per week for 78 weeks was associated with a significantly increased

incidence of alveolar/bronchiolar adenoma and carcinoma (combined) in high-dose (300

mg/kg) animals, and a significant positive dose-response trend was observed (NCI

1979a). NCI questioned the significance of these lung tumors because the incidence in

the control group was unusually low compared with historical untreated controls, and

only small numbers of vehicle historical controls were available from the same testing

laboratory. [However, a larger number of vehicle (corn oil)-treated historical controls

from this same time period (prior to 1979), with similar study duration, and from the

same source as the styrene study were available from a different testing laboratory.

Results from these historical vehicle controls indicated that the concurrent vehicle

controls in the NCI study were not unusually low and the lung tumor incidence in the

high-dose group was significantly increased compared with those historical controls.]

There also was a significant dose-response trend for hepatocellular adenomas in female

B6C3F1 mice, but no significant pair-wise comparisons were observed. The other gavage

study included a single dose of styrene administered to pregnant dams on gestation day

17 and weekly exposures of the pups after weaning (Pomomarkov and Tomatis 1978).

O20 mice (a strain with a high spontaneous incidence of lung tumors) were dosed at

1,350 mg/kg and C57Bl mice were dosed at 300 mg/kg. A significantly higher incidence

and earlier onset of lung tumors (adenoma and carcinoma combined) occurred in both

male and female O20 mice compared with vehicle controls. Tumor incidence was not

significantly increased in C57Bl mice.


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Significantly increased incidences of alveolar/bronchiolar adenoma and

alveolar/bronchiolar adenoma or carcinoma (combined) occurred in male CD-1 mice at

inhalation exposure concentrations of 40 to 160 ppm over a period of 104 weeks and in

female mice at exposure concentrations of 20, 40, and 160 ppm over a period of 98 weeks

(Cruzan et al. 2001). Female mice in the high-dose (160-ppm) group also had increased

incidences of alveolar/bronchiolar carcinoma.

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No increased incidences of tumors were observed in female A/J mice (also a strain

susceptible to lung tumors) treated with 20 intraperitoneal injections of styrene over 7

weeks (total dose of 200 μmol [approximately 100 mg/kg b.w.]) and evaluated 20 weeks

after the last injection (Brunnemann et al. 1992).

Rats

Several of the studies in rats were limited because of short duration, high mortality,

incomplete histopathology, or incomplete reporting. None of the carcinogenicity studies

reviewed in rats showed evidence of lung tumors, and none of the gavage (NCI 1979a,

Pomomarkov and Tomatis 1978, Conti et al. 1988), or intraperitoneal or subcutaneous

injection studies (Conti et al. 1988) reported an increased incidence in any tumor type.

An oral gavage study in F344 rats (NCI 1979a) and an inhalation study in Sprague-

Dawley rats (Cruzan et al. 1998) were the most robust and most completely reported

carcinogenicity studies. Neither study showed an increase in tumor incidences in styrene-

exposed rats, although Sprague-Dawley rats exhibited a negative trend in pituitary and

mammary gland tumors and a positive trend for testicular interstitial-cell tumors. In

another inhalation study in Sprague-Dawley rats, there was a dose-related increase in the

incidences of malignant mammary gland tumors; treatment-related and statistically

significant incidences of these tumors were seen in the top three dose groups (Conti et al.

1988). A drinking-water study did not report any dose-related carcinogenic effects

(Beliles et al. 1985). However, statistical reanalyses of study data indicated a marginal

increase in the incidence of mammary fibroadenoma in high-dose female rats and a

significant dose-related trend. Another inhalation study (Jersey et al. 1978) [unpublished

but reviewed in several published reports] indicated that styrene was associated with a


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statistically significant increase in incidence of mammary adenocarcinoma in the low-

(600-ppm) but not high-dose (1000-ppm) group and a significant increase (when

compared with historical but not concurrent controls) in the combined incidence of

lymphosarcoma and leukemia in female rats in both the 600-ppm and 1000-ppm dose

groups. The authors did not consider the mammary adenocarcinomas to be causally

associated with styrene exposure because the incidence of mammary adenocarcinoma

was low compared with historical controls and there was no incidence of mammary

adenocarcinoma in the high-dose group. Elevated incidences of leukemia/lymphosarcoma

were observed in both treatment groups of female Sprague Dawley rats in this inhalation

study.

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Mixtures and Metabolite Studies

No increase in tumor incidence was observed in rats exposed by gavage (3 days per

week) to a mixture of 70% styrene and 30% β-nitrostyrene over 78 weeks (NCI 1979b),

but an increased incidence of lung tumors was observed in male mice in the 175 mg/kg

dose group, but not in the 350 mg/kg dose group exposed to this styrene/β-nitrostyrene

mixture. [However, because of poor survival of the high-dose male mice there were

substantially fewer animals at risk for late-occurring tumors.]

The styrene metabolite, styrene-7,8-oxide, was previously evaluated for carcinogenicity

and is listed in the Report on Carcinogens [first listed in the 10th Report on Carcinogens,

2002] as reasonably anticipated to be a human carcinogen based on forestomach tumors

in rats and mice and liver tumors in male mice.

Absorption, Distribution, Metabolism, and Excretion

Styrene can be absorbed through inhalation, ingestion, or skin contact, but the most

important route of exposure in humans in occupational settings is by inhalation, which

results in rapid absorption and distribution of approximately 60% to 70% of inhaled

styrene; the highest tissue concentrations are in subcutaneous fat. Food is also an

important source of exposure for the general population. Metabolic activation of styrene

results in formation primarily of the genotoxic metabolite styrene-7,8-oxide, which can

be detoxified by glutathione conjugation or conversion to styrene glycol by microsomal


9/29/08 xvii

epoxide hydrolase. Styrene is metabolized in both the liver and the lung, and the Clara

cells in the lung are regarded as the major cell type in styrene activation following

inhalation exposure. The initial step in styrene metabolism is catalyzed by cytochromes

P450; CYP2E1 and Cyp2f2 are the predominant enzymes in humans and experimental

animals. In animals, CYP2E1 predominates in liver, while Cyp2f2 is the primary enzyme

in mouse lung. CYP2A13, CYP2F1, CYP2S1, CYP3A5, and CYP4B1 are preferentially

expressed in the lung compared with liver in humans, and the human CYP2F1 has been

shown to be capable of metabolizing styrene when expressed in vitro. Because styrene-

7,8-oxide contains a chiral carbon, this and some subsequent styrene metabolites can

exist as either R- or S-enantiomers. A second metabolic pathway through styrene-3,4-

oxide results in formation of 4-vinylphenol, which has been detected in humans, rats, and

mice in vivo, but the importance of 4-vinylphenol in styrene toxicity has not been well

characterized. Almost all absorbed styrene is excreted as urinary metabolites, primarily

mandelic acid and phenylglyoxylic acid.

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Species differences exist among rats, mice, and humans in the metabolism and toxicity of

styrene, which may be related, at least in part, to interspecies differences in the

stereochemistry of metabolism. The R-enantiomer, which has been suggested by some

reports to be more toxic than the S-form, has been reported to be produced in relatively

larger amounts in mouse lung than in rat lung, but the difference was less pronounced

when microsomal preparations were used. In mice, the R-isomer of styrene-7,8-oxide was

significantly more hepatotoxic than the S-isomer; the toxicity of the R-isomer also was

greater in the lung, but the difference was not statistically significant.

Toxicity

Styrene exposure has been associated with numerous health effects in humans and

laboratory animals. The acute toxicity of styrene is low to moderate with an oral LD50 of

320 mg/kg and an inhalation LC50 of 4,940 ppm (4-hour exposure) in mice and an oral

LD50 of 5,000 mg/kg and an inhalation LC50 of 2,770 ppm (2-hour exposure) in rats. The

primary effects of acute exposure to styrene in experimental animals and humans include

irritation of the skin, eyes, and respiratory tract and CNS effects. Drowsiness, listlessness,


xviii 9/29/08

muscular weakness, and unsteadiness are common signs of systemic styrene intoxication.

Several studies have reported effects on color vision, hearing threshold, reaction time,

and postural stability following long-term occupational exposure to styrene at

concentrations ranging from about 20 to 30 ppm. Reports of ischemic heart disease and

hepatic, renal, hematological, and immunological effects have been inconsistent. Human

data are insufficient to determine whether styrene is a reproductive or developmental

toxicant, but effects of styrene to increase serum prolactin levels in humans have been

reported.

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Styrene toxicity in experimental animals is similar to that reported in humans. Exposure

to styrene vapors can cause eye and respiratory tract irritation, CNS depression, and

death. Clara cells are the main target of styrene pneumotoxicity, and the available data

indicate increased susceptibility in the mouse. Glutathione depletion as a result of styrene

exposures has been reported to be associated with damage to lung, liver, and kidney

tissues. The cytotoxicity of styrene in the mouse lung, a tissue high in CYP2F isoforms,

could be prevented by CYP2F inhibitors. Some studies have reported reproductive and

developmental effects, but these effects were seen mostly at doses associated with

maternal toxicity. Reported effects have included embryonic, fetal, and neonatal death,

skeletal and kidney abnormalities, decreased birth weight, neurobehavioral abnormalities,

and postnatal developmental delays. The possibility of polystyrene dimer and trimer

extracts from food containers mimicking the physiological effects of estrogen have also

been investigated, but with a mixture of positive and negative results.

Genetic Damage

In vitro studies show that styrene-7,8-oxide forms DNA adducts and causes single-strand

breaks in a dose-related manner. Several studies have shown a correlation between

single-strand breaks and DNA adducts and indicate that the strand breaks, which are not

generally regarded as significantly lethal or mutagenic lesions, are efficiently repaired

within several hours after exposure has stopped. Adducts are formed primarily at the N7-,

N2-, and O6-positions of guanine. N7-adducts are formed in the greatest amount but are

the least persistent, while O6-adducts are formed in the least amount but are the most

persistent. Styrene-7,8-oxide was mutagenic without metabolic activation in all in vitro


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mutagenicity test systems reported and caused mutations in some studies in the presence

of metabolizing enzymes. Both styrene and styrene-7,8-oxide caused cytogenetic effects

(sister chromatid exchange [SCE], chromosomal aberrations, and micronuclei) in human

lymphocytes or other mammalian cells in vitro. DNA adducts have been detected in liver

and lung cells of mice and rats exposed to styrene in vivo, although the levels varied

across studies. The majority of studies in experimental animals demonstrated an effect of

both styrene-7,8-oxide and styrene exposure on single-strand breaks, while both positive

and negative results for cytogenetic or clastogenic effects of styrene were reported.

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DNA adducts, primarily N7- and O6-adducts, were reported in white blood cells in all

studies of styrene-exposed workers employed mainly in hand-lamination plants. In most

studies in workers, single-strand breaks showed exposure-related increases; however, two

studies gave negative results. The limited data on mutation frequencies in HPRT and

GPA in styrene-exposed workers are inconclusive. More than half the studies measuring

chromosomal aberrations have reported an increase in chromosomal aberrations in

styrene-exposed workers (or subgroups of workers), and several studies have reported a

positive exposure-response relationship with styrene air levels or urinary metabolites. A

meta-analysis of 22 studies found a positive association between styrene exposure level

and chromosomal aberration frequency when exposure levels were dichotomized as

greater than or less than a threshold value of 30 ppm for an 8-hour time-weighted

average. Studies of other cytogenetic markers in humans are conflicting. About half of

the studies that evaluated micronucleus and SCE frequency in styrene workers were

positive, and a few studies have reported significant dose-response relationships with

styrene exposure. A meta-analysis of 10 micronucleus studies was inconclusive, and a

meta-analysis of 14 SCE studies indicated a slight increase in SCE frequency but, again,

was too small to be conclusive. A number of studies have been published on the possible

modulating role of genetic polymorphisms, mainly in xenobiotic metabolism enzymes

and DNA-repair genes, at the level of various biomarkers. Some authors have suggested

that genetic susceptibility (probably at many loci) may be important in styrene-mediated

genotoxicity.


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Mechanistic Data 1

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The proposed mechanisms for the carcinogenicity of styrene include both genotoxic and

epigenetic pathways. These mechanisms, which are not necessarily mutually exclusive,

include: (1) metabolic conversion of styrene to styrene-7,8-oxide and subsequent

induction of DNA damage in the target tissue and (2) cytotoxic effects of styrene

metabolites in the mouse lung. A variety of DNA adducts (including some at base-pairing

sites on nucleotides) induced by styrene and styrene-7,8-oxide has been identified in

human cells, experimental animals, and occupationally exposed workers, but the covalent

binding indices for both molecules are relatively low in rats and mice. The DNA damage

induced by styrene exposure, including single-strand breaks, was found to correlate

significantly with markers of styrene exposure in some studies of styrene workers.

Styrene is mutagenic through the formation of styrene-7,8-oxide (in vitro). A number of

studies reported a positive association between occupational exposure to styrene and the

frequency of chromosomal aberrations, with some studies reporting exposure-response

relationships. Some authors have suggested that polymorphisms in DNA-repair genes

could put some individuals at higher risk for styrene genotoxicity or carcinogenicity.

Many researchers have tried to explain why lung tumors were observed in mice but not in

rats in long-term inhalation exposure studies. Some researchers have proposed that

styrene exposure causes pulmonary hyperplasia in the mouse lung, which may play a role

in the development of lung tumors. Effects of repeated styrene exposure observed in the

lungs of mice, but not in rats, included focal crowding of bronchiolar cells, bronchiolar

epithelial hyperplasia, and bronchiolo-alveolar hyperplasia. The Harvard Center for Risk

Analysis (Cohen et al. 2002) considered three factors as possible explanations for the

greater susceptibility of mouse lung than rat lung to development of hyperplasia leading

to tumors with exposure to styrene are: (1) the presence of the styrene-metabolizing

cytochromes in mouse lung tissues, (2) greater formation of the R-enantiomer of styrene-

7,8-oxide, and (3) the susceptibility of mouse lung tissue to glutathione depletion.

However, they concluded that although toxicokinetic models generally predict higher

rates of metabolism by mice and rats than by humans, the models do not consistently

predict a difference between the rodent species. An alternative mechanism is that


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interspecies differences in styrene toxicity are most likely explained through CYP2F-

generated metabolites (2f2 in mice, 2F4 in rats, and 2F1 in humans) in the mouse lung.

This is based on data showing that most of the effects of cytotoxicity and tumor

formation were seen in mouse respiratory tissues, which are high in CYP2F isoforms, and

that CYP2F inhibitors prevented cytotoxicity. Moreover, metabolites formed from ring

oxidation, including 4-vinylphenol, are about 6-fold higher in mice compared with rats,

and 4-vinylphenol is more potent than styrene-7,8-oxide as a pneumotoxicant.

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Abbreviations ABS: acrylonitrile-butadiene-styrene

ACGIH: American Conference of Governmental Industrial Hygienists

ADH: alcohol dehydrogenase

ALDH: aldehyde dehydrogenase

AlO: aldehyde oxidase

ALL: acute lymphocytic leukemia

AML: acute myelogenous leukemia

ANOVA: analysis of variance

ASPEN: Assessment System for Population Exposure Nationwide

ATSDR: Agency for Toxic Substances and Disease Registry

BCF: bioconcentration factor

BEAM: Boston Exposure Assessment in Microenvironments

BEI: biological exposure indices

BLS: Bureau of Labor Statistics

BRCA1: breast cancer 1, early onset gene

b.w.: body weight

C: control

C+: centromere positive

C–: centromer negative

CA: chromosomal aberrations

Cal/OSHA: California Division of Occupational Safety and Health

CBI: covalent binding index

CC1b: Clara-cell specific protein

CDC: Centers for Disease Control and Prevention


CEH: Chemical Economics Handbook

CERHR: Center for Evaluation of Risks to Human Reproduction

CHO: Chinese hamster ovary

CLL: chronic lymphocytic leukemia

cm: centimeter

CML: chronic myeloid leukemia

CNS: central nervous system

CO: cyclohexene oxide

CPBI: cytokinesis proliferation block index

CR: creatinine

CREST: calcinosis-Raynaud’s phenomenon-oesophageal dismobility-sclerodactyly-telangiectasis syndrome of scleroderma

CYP: cytochrome P450

Cyt-B: cytochalasin B

d: day

Da: Dalton

DAPI: 4',6-diamidino-2-phenylindol·2HCl

DC: decarboxylase

dm: decimeter

DMDTC: dimethyldithiocarbamate

DMSO: dimethylsulfoxide

DNA: deoxyribonucleic acid

DOT: Department of Transportation

E: exposed

EPA: Environmental Protection Agency

EPHX: epoxide hydrolase

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9/29/08 xxv

ETS: environmental tobacco smoke

E.U.: European Union

F: female

FDA: Food and Drug Administration

FISH: fluorescence in-situ hybridization

g: gram

GGT: gamma-glutamyl transpeptidase

GI: gastrointestinal

GPA: glycophorin A

GSH: glutathione

GSTM1: glutathione S transferase M1

GSTT1: glutathione S transferase T1

γ-GT: gammaglutamyl transpeptidase

h: hour

HA: hydroxylapatite

HazDat: Hazardous Substances Release and Health Effects Database

HE: human erythrocytes

HEL: human embryonic lung

HFC: high-frequency cells

HIC: highest ineffective concentration

HID: highest ineffective dose

HPRT: hypoxanthine phosphoribosyltransferase

HSDB: Hazardous Substances Data Bank

Hz: Hertz

IARC: International Agency for Research on Cancer


ICD: International Classification of Diseases

i.p.: intraperitoneal

IRR: incidence rate ratio

JEM: job-exposure matrix

K+: kinetochore-positive

kg: kilogram

Koc: soil organic carbon-water partitioning coefficient

Kow: octanol-water partition coefficient

L: liter

LC: liquid chromatography

LD50: lethal dose for 50% of the population

LEC: lowest effective concentration

LED: lowest effective dose

LH: lymphohematopoietic

LHC: lymphohematopoietic cancer

LWAE: lifetime weighted average exposure

M: male

m3: cubic meter

MA: mandelic acid

mEH: microsomal epoxide hydrolase

mfg.: manufacturing

mg: milligram

mL: milliliter

MM: multiple myeloma

MN: micronuclei

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MNBC: binucleated lymphocytes

MNMC: mononucleated lymphocytes

mol wt: molecular weight

MS: mass spectrometry

N: sample size

NA: not available

NA-AAF: N-acetoxy-2-acetylaminofluorene

NAcT: N-acetyltransferase

NADPH: nicotinamide adenine dinucleotide phosphate, reduced form

NAP: not applicable

NCEs: micronucleated normochromatic erythrocytes

NCHS: National Center for Health Statistics

NCI: National Cancer Institute

ND: not detected

NDMA: N-nitrosodimethylamine

NDT: not determined

NHANES: National Health and Nutrition Examination Survey

NHL: non-Hodgkin’s lymphoma

NI: not identified

NIEHS: National Institute of Environmental Health Sciences

NIOSH: National Institute for Occupational Safety and Health

ng: nanogram

NLM: National Library of Medicine

NNK: 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone

No.: number


NQ: not quantified

NR: not reported

NRC: National Response Center

NS: not significant

NT: not tested

NTP: National Toxicology Program

OH: hydroxyl

OR: odds ratio

OSHA: Occupational Safety and Health Administration

PAH: polycyclic aromatic hydrocarbon

PAMA: phenacylmercapturic acid

PBL: peripheral blood lymphocytes

PBPK: physiologically based pharmacokinetic model

PC: personal computer

PCEs: micronucleated polychromatic erythrocytes

PEL: permissible exposure limit

PGA: phenylglyoxylic acid

PHA: phytohemagglutinin

PHEMA: phenylhydroxyethyl mercapturic acids

PWN: pokeweed

ppb: parts per billion

ppbv: parts per billion by volume

ppm: parts per million

r: correlation coefficient

REL: recommended exposure limit

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RoC: Report on Carcinogens

RR: relative risk

RTECS: Registry of Toxic Effects of Chemical Substances

RV: recreational vehicle

s.c.: subcutaneous

SB: styrene in blood

SBR: styrene-butadiene rubber

SCE: sister chromatid exchange

SD: standard deviation

SDH: sorbitol dehydrogenase

SE: standard error of the mean

SIR: standardized incidence ratio

SIRC: Styrene Information and Research Center

SO: styrene oxide

SOC: Standard Occupational Classification

SOCMI: Synthetic Organic Chemical Manufacturing Industry

SSB: single-strand breaks

STEL: short-term exposure limit

TDS: Total Diet Study

TK: thymidine kinase

TLV: threshold-limit value

TRI: Toxics Release Inventory

TWA: time-weighted average

UB: styrene in urine

UDS: unscheduled DNA synthesis


USITC: United States International Trade Commission

μg: microgram

VOC: volatile organic chemical

VPT: vinylphenol

WHO: World Health Organization

XO: xanthine oxidase

XPC: xeroderma pigmentosum, complementation group C

XPD: xeroderma pigmentosum, complementation group D

XPG: xeroderma pigmentosum, complementation group G

XRCC: X-ray repair cross-complementing group

yr: year

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Table of Contents

1 Introduction............................................................................................................................... 1 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

1.1 Chemical identification.............................................................................................. 1 1.2 Physical-chemical properties ..................................................................................... 2 1.3 Metabolites................................................................................................................. 3 1.4 Analogues .................................................................................................................. 7

2 Human Exposure....................................................................................................................... 9 2.1 Use ............................................................................................................................. 9 2.2 Production................................................................................................................ 11 2.3 Environmental release, fate, and occurrence ........................................................... 14

2.3.1 Air ............................................................................................................. 16 2.3.2 Water......................................................................................................... 27 2.3.3 Soil............................................................................................................ 30 2.3.4 Food .......................................................................................................... 32

2.4 General population exposure ................................................................................... 38 2.5 Occupational exposure............................................................................................. 44

2.5.1 The reinforced plastics industry................................................................ 45 2.5.2 The styrene-butadiene rubber (SBR) industry.......................................... 61 2.5.3 Styrene-butadiene rubber production exposure levels.............................. 65 2.5.4 The styrene monomer and polymer industry ............................................ 68 2.5.5 Other occupational exposures................................................................... 72

2.6 Biological indices of exposure................................................................................. 74 2.7 Regulations and guidelines ...................................................................................... 81

2.7.1 Regulations ............................................................................................... 81 2.7.2 Guidelines ................................................................................................. 82

2.8 Summary .................................................................................................................. 83 3 Human Cancer Studies............................................................................................................ 85

3.1 The reinforced-plastics industry .............................................................................. 87 3.1.1 Washington state....................................................................................... 87 3.1.2 United Kingdom ....................................................................................... 90 3.1.3 United States............................................................................................. 91 3.1.4 Denmark ................................................................................................... 93 3.1.5 Denmark, Finland, Norway, Italy, Sweden, and the United

Kingdom. .................................................................................................. 96 3.2 The styrene-butadiene rubber industry .................................................................. 108

3.2.1 United States: McMichael et al. ............................................................. 109 3.2.2 United States: Meinhardt et al. ............................................................... 109 3.2.3 United States and Canada: Matanoski, Santos-Burgoa, and

coworkers................................................................................................ 110 3.2.4 United States and Canada: Delzell, Sathiakumar, Macaluso, Graff ....... 113


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3.3 The styrene monomer and polymer industry ......................................................... 136 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

3.3.1 Germany ................................................................................................. 136 3.3.2 United States- multi-plant....................................................................... 137 3.3.3 United States- single plant ...................................................................... 138 3.3.4 United Kingdom ..................................................................................... 139

3.4 Other cohort studies ............................................................................................... 142 3.4.1 Styrene-exposed workers (biomarker study) .......................................... 142 3.4.2 Environmental exposure ......................................................................... 142

3.5 Case-control and ecological studies....................................................................... 146 3.5.1 Lymphohematopoietic cancers ............................................................... 146 3.5.2 Breast cancer........................................................................................... 147 3.5.3 Series of studies in a Canadian population............................................. 149 3.5.4 Lung cancer and styrene exposure.......................................................... 150

3.6 [Strengths and limitations of the literature] ........................................................... 156 3.6.1 Utility of the studies................................................................................ 156 3.6.2 Misclassification of disease and exposure.............................................. 158 3.6.3 Other possible biases and confounding .................................................. 161

3.7 Summary of previous evaluations (IARC and Cohen et al.) ................................. 164 3.8 Summary of the findings for selected cancer sites................................................. 165

3.8.1 Esophageal cancer .................................................................................. 173 3.8.2 Pancreatic cancer .................................................................................... 174 3.8.3 Laryngeal cancer..................................................................................... 175 3.8.4 Lung cancer ............................................................................................ 175 3.8.5 Lymphohematopoietic cancers ............................................................... 176 3.8.6 Other sites ............................................................................................... 183

3.9 Summary ................................................................................................................ 188 4 Studies of Cancer in Experimental Animals......................................................................... 195

4.1 Mice ....................................................................................................................... 195 4.1.1 Oral ......................................................................................................... 196 4.1.2 Inhalation ................................................................................................ 200 4.1.3 Intraperitoneal injection.......................................................................... 202

4.2 Rats ........................................................................................................................ 202 4.2.1 Oral ......................................................................................................... 202 4.2.2 Inhalation ................................................................................................ 207 4.2.3 Parenteral administration ........................................................................ 211

4.3 Mixtures containing styrene................................................................................... 212 4.4 Styrene metabolites................................................................................................ 213 4.5 Summary ................................................................................................................ 214

5 Other Relevant Data.............................................................................................................. 221 5.1 Absorption, distribution, metabolism, and excretion............................................. 221


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5.1.1 Absorption .............................................................................................. 221 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

5.1.2 Distribution............................................................................................. 223 5.1.3 Metabolism ............................................................................................. 225 5.1.4 Excretion................................................................................................. 238

5.2 Toxicity .................................................................................................................. 240 5.2.1 Humans ................................................................................................... 240 5.2.2 Experimental animals ............................................................................. 246 5.2.3 Estrogenicity studies............................................................................... 256

5.3 Interspecies differences in metabolism, toxicity, and toxicokinetics .................... 258 5.3.1 Styrene-7,8-oxide formation in the lung................................................. 258 5.3.2 Detoxification of styrene-7,8-oxide in respiratory tissue ....................... 259 5.3.3 Stereochemistry considerations .............................................................. 260 5.3.4 Kinetics of styrene metabolism and toxicokinetic models ..................... 262

5.4 Genetic and related effects..................................................................................... 264 5.4.1 DNA adduct formation ........................................................................... 264 5.4.2 In vitro studies ........................................................................................ 268 5.4.3 In vivo studies in experimental animals.................................................. 278 5.4.4 Studies in styrene-exposed workers........................................................ 289 5.4.5 Genetic polymorphisms and susceptibility to styrene-mediated

genotoxicity ............................................................................................ 360 5.4.6 Summary of styrene and styrene-7,8-oxide genotoxicity ....................... 366

5.5 Mechanistic studies and considerations ................................................................. 368 5.5.1 Genotoxicity ........................................................................................... 370 5.5.2 Gene expression and apoptosis............................................................... 373 5.5.3 Oxidative stress....................................................................................... 374 5.5.4 Cytotoxic effects of styrene on mouse lung ........................................... 375 5.5.5 Selected styrene analogues ..................................................................... 377

5.6 Summary ................................................................................................................ 380 5.6.1 Absorption, distribution, metabolism, and excretion.............................. 380 5.6.2 Toxicity................................................................................................... 380 5.6.3 Interspecies differences in metabolism, toxicity, and toxicokinetics ..... 381 5.6.4 Genetic and related effects...................................................................... 382 5.6.5 Mechanistic studies and considerations.................................................. 383

6 References............................................................................................................................. 385 Glossary of Terms....................................................................................................................... 455

List of Tables

Table 1-1. Chemical identification of styrene................................................................................. 2 Table 1-2. Physical and chemical properties of styrene ................................................................. 3


Table 1-3. Urinary metabolites of styrene in rodents and humans ................................................. 5 Table 1-4. Styrene analogues.......................................................................................................... 7 Table 2-1. Styrene use in industrial resin...................................................................................... 10 Table 2-2. Residual styrene-monomer levels in polymer and copolymer materials in 1980 ....... 18 Table 2-3. Historical levels of residual styrene (mg/kg) in polymer and copolymer: 1976–

1980............................................................................................................................. 19 Table 2-4. Concentrations of styrene in outdoor air in the United States..................................... 22 Table 2-5. U.S. levels of styrene measured in indoor air and by personal monitoring................. 26 Table 2-6. Levels of styrene measured in U.S. waters.................................................................. 29 Table 2-7. Measurements of styrene in foods packaged in polystyrene ....................................... 34 Table 2-8. Food levels of styrene [source of styrene unknown]................................................... 36 Table 2-9. Summary of styrene levels in FDA’s Total Diet Study (1991–2003a)........................ 38 Table 2-10. Daily styrene intakes for the general public from various sources ........................... 39 Table 2-11. Estimated daily intake of styrene from various media for Canadians of

different ages............................................................................................................... 40 Table 2-12. Estimated annual and lifetime exposures for the general public............................... 42 Table 2-13. Summary of measured styrene exposure levels in the reinforced plastics

industry. ...................................................................................................................... 55 Table 2-14. Summary of occupational styrene exposure levels in the styrene-butadiene

rubber industry ............................................................................................................ 67 Table 2-15. Summary of occupational styrene exposure levels in the styrene monomer

and polymer industry in the United States.................................................................. 71 Table 3-1. Epidemiologic studies of cancer risk following styrene exposure in the

reinforced-plastics industry, 1985–2004 (results of the most recent follow-upa)....... 99 Table 3-2. Risk of leukemia with cumulative and peak exposurea to styrene, butadiene,

and DMDTCb ............................................................................................................ 123 Table 3-3. Cumulative exposure to styrene, butdadiene and DMDTC and risk of NHL

and NHL+CLL.......................................................................................................... 126 Table 3-4. Epidemiologic studies of cancer risk following styrene exposure in the

styrene-butadiene rubber industry, 1976–2005......................................................... 127 Table 3-5. Cohort studies of cancer risk following styrene exposure in the styrene

monomer and polymer industry, 1978–1992 ............................................................ 140 Table 3-6. Other cohort studies evaluating cancer risk and exposure to styrene........................ 144 Table 3-7. Case-control and ecological studies evaluating cancer risk and exposure to

styrene ....................................................................................................................... 151 Table 3-8. Relative occurrence of cancer in 12 cohort studies of populations exposed to

styrene (total study populations)............................................................................... 168 Table 3-9. Mortality or incidence of selected cancers among all workers in the reinforced-

plastics industry ........................................................................................................ 186

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Table 3-10. Mortality or incidence of selected cancers among workers in high-styrene–exposure groups (laminators and others)* in the reinforced-plastics industry ......... 187

Table 4-1. Tumor incidences in B6C3F1 mice exposed to styrene by gavage for 78 weeks and surviving for at least 52 weeks........................................................................... 198

Table 4-2a. Lung tumor incidences in O20 mice exposed to styrene in utero and weekly by gavage for 16 weeks after weaning...................................................................... 199

Table 4-2b. Tumor incidences in C57Bl mice exposed to styrene in utero and weekly by gavage for 120 weeks after weaning......................................................................... 199

Table 4-3. Lung tumor incidence in CD-1 mice exposed to styrene by inhalation for 98 or 104 weeksb ................................................................................................................ 201

Table 4-4. Mammary gland tumor incidence in Sprague-Dawley rats exposed to styrene in drinking water for 104 weeks ............................................................................... 205

Table 4-5. Summary of carcinogenicity studies in rats exposed to styrene by oral administration ........................................................................................................... 206

Table 4-6. Incidence of mammary tumors in Sprague-Dawley rats exposed to styrene by inhalation for 52 weeks............................................................................................. 208

Table 4-7. Mammary tumors and leukemia or lymphosarcoma in Sprague-Dawley rats exposed to styrene by inhalation for 18 to ~21 months ............................................ 210

Table 4-8. Tumor incidences in Sprague-Dawley rats exposed to styrene by inhalation for 104 weeks.................................................................................................................. 211

Table 4-9. Tumor incidences in B6C3F1 mice exposed to a mixture of β-nitrostyrene and styrene for 79 weeks ................................................................................................. 213

Table 4-10. Summary of neoplastic lesions in mice and rats exposed to styrene-7,8-oxide by gavage .................................................................................................................. 214

Table 4-11. Summary of studies in mice .................................................................................... 217 Table 4-12. Summary of studies in ratsa ..................................................................................... 218 Table 5-1. Production of R- and S-enantiomers of styrene-7,8-oxide by cell preparations

enriched in either Clara cells or type II cells from rat and mouse lungsa ................. 229 Table 5-2. Styrene-7,8-oxide DNA adducts formed in mammalian cells in vitro ...................... 269 Table 5-3. DNA damage in mammalian cells exposed to styrene-7,8-oxide ............................. 272 Table 5-4. Mutagenicity of styrene and styrene-7,8-oxide in vitro ............................................ 274 Table 5-5. Cytogenetic effects of styrene in vitro....................................................................... 277 Table 5-6. Cytogenetic effects of styrene-7,8-oxide in vitro, without metabolic activation ...... 279 Table 5-7. Formation of styrene-7,8-oxide DNA adducts in animals exposed to styrene.......... 282 Table 5-8. DNA damage in experimental animals exposed to styrene or styrene-7,8-

oxide.......................................................................................................................... 286 Table 5-9. Cytogenetic effects of styrene and styrene-7,8-oxide in experimental animals........ 289 Table 5-10. Studies of DNA adducts in white blood cells of workers occupationally

exposed to styrene in Bohemia, the United States, and Germany ............................ 293


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Table 5-11. DNA damage (single-strand breaks or alkali-labile sites) in workers occupationally exposed to styrene ............................................................................ 299

Table 5-12. Mutation frequencies in workers exposed to styrene .............................................. 306 Table 5-13. Chromosomal aberrations in lymphocytes from workers occupationally

exposed to styrene..................................................................................................... 309 Table 5-14. Micronuclei in lymphocytes from workers occupationally exposed to styrene ...... 332 Table 5-15. Sister chromatid exchange in lymphocytes from workers occupationally

exposed to styrene..................................................................................................... 347 Table 5-16. Genotype analyses in in vitro studies with styrene and styrene-7,8-oxide.............. 362 Table 5-17. Genotype analyses in vivo in workers occupationally exposed to styrene in

association with biomarkers of genotoxicity ............................................................ 365 Table 5-18. Genetic and related effects of styrene ..................................................................... 367

List of Figures

Figure 1-1. Chemical structures of styrene and polystyrene........................................................... 1 Figure 1-2. Stereoisomers of styrene-7,8-oxide (epoxyethylbenzene) ........................................... 4 Figure 2-1. Synthesis of styrene from ethylbenzene and polymerization of styrene to form

polystyrene.................................................................................................................. 12 Figure 2-2. U.S. styrene production (1960–2006) ........................................................................ 13 Figure 2-3. U.S. imports and exports for styrene.......................................................................... 14 Figure 2-4. Temporal decline in styrene exposure scores (short-term samples [< 1 h])

estimated for reinforced plastics workers ................................................................... 48 Figure 2-5. Temporal decline in styrene exposure scores short-term samples [< 1 h])

estimated for reinforced plastics workers ................................................................... 49 Figure 2-6. Typical continuous emulsion styrene-butadiene rubber polymerization

process......................................................................................................................... 63 Figure 2-7. Typical emulsion styrene-butadiene rubber finishing process................................... 64 Figure 2-8. Solution styrene-butadiene rubber manufacture by continuous process.................... 65 Figure 2-9. Polymerization of polystyrene by the continuous process......................................... 69 Figure 5-1. Styrene metabolism in humans ................................................................................ 226 Figure 5-2. Pneumotoxicity and hepatotoxicity of styrene-7,8-oxide enantiomers in male

non-Swiss albino mice at 24 hours after i.p. administration..................................... 254 Figure 5-3. Styrene-7,8-oxide binding sites in DNA (from Vodicka et al. 2002a) .................... 266


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1 Introduction 1

Styrene is a very important monomer used worldwide in the production of polymers,

which are incorporated into products such as rubber, plastic, insulation, fiberglass, pipes,

automobile parts, food containers, and carpet backing. Most of these products contain

both free styrene monomer and styrene polymerized in long chains (polystyrene or mixed

polymers) (ATSDR 1992).

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Styrene was nominated for possible listing in the Report on Carcinogens by a private

individual based on its widespread use and exposure and evidence of carcinogenicity

from studies in humans and experimental animals. The International Agency for Research

on Cancer (IARC) currently classifies styrene as possibly carcinogenic to humans (Group

2B) based on limited evidence in humans and limited evidence in experimental animals

(IARC 1994a, 2002). Styrene-7,8-oxide, a major metabolite of styrene, has been

classified by the Report on Carcinogens as reasonably anticipated to be a human

carcinogen based on sufficient evidence of carcinogenicity in experimental animals (NTP

2004). IARC (1994b) also classifies styrene-7,8-oxide as probably carcinogenic to

humans (Group 2A) based on sufficient evidence in experimental animals (forestomach

tumors in rats and mice and liver tumors in male mice) and mechanistic data.

1.1 Chemical identification 18 Styrene is an aromatic hydrocarbon with the structure illustrated in Figure 1-1. Styrene

can polymerize to form polystyrene (Figure 1-1). Table 1-1 contains chemical

identification information for styrene.

Figure 1-1. Chemical structures of styrene and polystyrene


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Table 1-1. Chemical identification of styrene

Characteristic Styrene Styrene-7,8-oxide Chemical Abstracts index name

styrene phenyloxirane

CAS Registry number 100-42-5 96-09-3 Molecular formula C8H8 C8H8O Synonyms cinnamene, cinnamol, ethenylbenzene,

phenylethylene, styrol, styrolene, vinylbenzene, vinylbenzol, phenylethene, styrole, NSC 62785, cinnamenol, stirolo, styreen, styren, styrene monomer, styron, styropol, styropor, vinylbenzen, FEMA Number 3234, NCI-CO2200, UN 2055, IMO 3.3, Standard Transportation No. 49072 65 1.

epoxyethylbenzene; 1,2-epoxyethylbenzene; 1,2-epoxy-1-phenylethane; 1-phenyl-1,2-epoxyethane; epoxystyrene; NCI-C54977; NSC 637

Source: ChemIDPlus 2008a, 2008b, HSDB 2008a, 2008b, IARC 1994b.

1.2 Physical-chemical properties 1 Styrene is a colorless or yellowish, viscous liquid with a sweet, floral odor (HSDB

2008a). It has a flash point of 34°C (closed cup), lower explosive limit of 0.9% to 1.1%

(v/v), upper explosive limit of 6.1% to 6.8% (v/v), and an autoignition temperature of

490°C. Styrene is highly flammable and easily ignited by heat, sparks, or flames and its

vapors may form explosive mixtures with air due to the formation of peroxides. Styrene

may polymerize when contaminated by oxidizing agents, halides, or when heated, and it

emits acrid fumes upon decomposition (NSC 2004, SPA 2008). Usually styrene is

stabilized for safe storage, transport, and use by an inhibitor, commonly p-tert-

butylcatechol (HSDB 2008a). Typical impurities are ethylbenzene (85 ppm maximum),

polymer content (10 ppm maximum), p-tert-butylcatechol (10 to 15 ppm or 45 to 55

ppm), aldehydes (as benzaldehyde) (200 ppm), peroxides (as H2O2) (0.0015% by weight

or 100 ppm maximum), benzene (1 ppm maximum), sulfur (25 ppm maximum), and

chlorides (as chlorine) (50 ppm maximum). The physical and chemical properties of

styrene are summarized in Table 1-2.

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Polystyrene is a colorless solid with a melting point of 240°C and a relative density of

1.04 to 1.13 (NIOSH 2008). It is insoluble in water and has a flash point of 345°C to

360°C. When burned or heated above 300°C, polystyrene decomposes and releases toxic

fumes, including styrene.


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Table 1-2. Physical and chemical properties of styrene Property Information Molecular weight 104.2 Melting point (°C) –31 Boiling point (°C) 145 Specific gravity 0.906 at 20°C Solubility water at 25°C acetone, alcohol, carbon tetrachloride,

carbon disulfide, diethyl ether, ethanol, methanol, n-heptane, toludene

benzene, petroleum ether

310 mg/L at 25°C soluble very soluble

Octanol-water partition coefficient (log Kow)

2.95

Dissociation constant (pKa) NA Vapor pressure (mm Hg) 6.4 at 25°C Vapor density 3.6 (air = 1) Critical temperature (°C) 363.7 Henry’s law constant 0.00275 atm-m3/mol at 25°C Hydroxyl radical reaction rate constant 5.8 x 10-11 cm3/molecule-sec at 25°C Sources: HSDB 2008a, IARC 1994a.

1.3 Metabolites 1 This section provides a brief overview of styrene metabolism in mammals and identifies

the major and minor metabolites detected in humans and rodents exposed to styrene.

Section 5 provides a more detailed discussion of styrene metabolism.

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Metabolism of styrene to styrene-7,8-oxide by cytochromes CYP2E1, CYP2B6, and

CYP2A13 has been reported to be the primary metabolic pathway in humans (Fukami et

al. 2008, Manini et al. 2002b, Manini et al. 2002a); however, as discussed in Section

5.1.3, other cytochromes, including CYP2F1, have been shown to be able to convert

styrene to styrene glycol when expressed in vitro. In addition, another toxic metabolite of

styrene, 4-vinylphenol, has been detected in small amounts in rats and humans and is

postulated to result from a ring-oxidation reaction forming styrene-3,4-oxide as an

intermediate.

The primary metabolite, styrene-7,8-oxide, is an epoxide that exists in two enantiomeric

forms (stereoisomers, or chemical compounds with asymmetric centers whose molecules


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are nonsuperimposable mirror images): the R- and S-isomers (Figure 1-2). Epoxides are

oxygen-containing heterocyclic compounds that are highly reactive because of the strain

associated with the three-membered ring structure (Melnick 2002). The oxide forms are

further metabolized to styrene glycol (phenylethylene glycol) by microsomal epoxide

hydrolase. Styrene glycol is then oxidized by alcohol and aldehyde dehydrogenases to

form mandelic acid and phenylglyoxylic acid and their conjugates, the main urinary

metabolites. Known and hypothesized urinary styrene metabolites are shown in Table 1-

3.

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Figure 1-2. Stereoisomers of styrene-7,8-oxide (epoxyethylbenzene)

The stereoisomers of styrene-7,8-oxide are designated as R- or S- based on ordering the

molecule with the lowest priority atom (hydrogen) away from the viewer as designated

by the dashed line. The order of the remaining substituents from highest to lowest (i.e.,

oxygen, carbon in the benzene ring, carbon in CH2) is oriented either clockwise (R-

isomer) or counterclockwise (S-isomer). Also, the two carbons attached to the benzene

ring are identified as the α- and β-carbons based on their order of attachment to the ring.

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Table 1-3. Urinary metabolites of styrene in rodents and humans

Metabolite Molecular

weight Structure

mandelic acid 152

hydroxymandelic acid 168

phenylglyoxylic acid 150

phenylglycinea 151

2-(4-hydroxy-phenyl)ethanol 138

phenylhydroxyethyl mercapturic acidsb (PHEMAs): M1

283

PHEMAs: M2b 283

phenacylmercapturic acid (PAMA) 281


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weight Structure

styrene glycol sulfate 218

4-vinylphenol sulfate 200

styrene glycol glucuronide 314

4-vinylphenol glucuronide 296

Source: Linhart et al. 2000, Manini et al. 2002b. aManini et al. (2002b) reported that this metabolite is expected to be formed, but it has never been demonstrated in urine following styrene exposure. bTwo diasteroisomers of each PHEMA exist (see Figure 5-1).


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1.4 Analogues 1 Many chemical analogues of styrene exist. Table 1-4 shows the structures of four

analogues that are discussed in Section 5. Ethyl benzene is a chemical precursor for

styrene (see Section 2), and it and 1-phenylethanol are metabolites of styrene (see Figure

5-1). 3-Methylstyrene and 4-methylstyrene are often used as a mixture called

vinyltoluene.

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Table 1-4. Styrene analogues


weight Structure

Ethyl benzene 106.2

1-Phenylethanol 122.2

3-Methylstyrene 118.2

4-Methylstyrene 118.2

ChemIDPlus 2008c, 2008d, 2008e, 2008f.



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2 Human Exposure 1

Styrene is used primarily in the manufacture of polystyrene; it is also used to manufacture

styrene-butadiene latex, styrene-butadiene rubber, unsaturated polyester resins, and

numerous other copolymers. The primary sources of exposure to the general public

include inhalation (including inhalation of indoor and outdoor ambient air, active

smoking, and exposure from environmental tobacco smoke), and ingestion of foods.

Workers may be exposed to high levels of styrene through inhalation and dermal

exposure. The industries with the largest numbers of highly-exposed workers are the

reinforced-plastics , styrene-butadiene rubber, and styrene monomer and polymer

industries. This section describes data important in evaluating human exposure to styrene,

including uses (Section 2.1), production (Section 2.2), the release, chemical fate, and

levels of styrene in various environmental media (Section 2.3), general population

exposures (Section 2.4), occupational exposures (Section 2.5), biological indices of

exposure (Section 2.6), and U.S. regulations and guidelines that are intended to reduce

exposure to styrene (Section 2.7). A summary of the human exposure section is provided

in Section 2.8.

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The information reported in this section was obtained from several peer-reviewed panel

evaluations or reviews for styrene, and from literature published since these reviews or

evaluations. The most recent panel evaluations include (1) an IARC monograph for

styrene (1994a, 2002), (2) the NTP’s Center for the Evaluation of Risks to Human

Reproduction (CERHR) Expert Panel Report on the Reproductive and Developmental

Toxicity of Styrene by Luderer et al. (2005), (3) the Evaluation Of The Potential Health

Risks Associated With Occupational And Environmental Exposure To Styrene by the

Harvard Center for Risk Analysis authored by Cohen et al. (2002) [sponsored by the

Styrene Information and Research Center], (4) the European Union’s 2002 Risk

Assessment Report for styrene, and (5) the ATSDR Toxicological Profile for Styrene

(1992).

2.1 Use 28 IARC (2002) reported that styrene was first isolated in 1831 through the distillation of a

natural balsam called storax. Styrene did not become commercially important, however,


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until World War II when the United States initiated a major program to develop synthetic

rubber (Miller et al. 1994, Steele et al. 1994). Since that time, styrene has become an

important chemical used in the synthesis and manufacture of polystyrene and hundreds of

different copolymers, as well as numerous other industrial resins (Guest 1997). Styrene

producers sell styrene monomer to companies (resin manufacturers and compound

producers) who use the styrene to make resins. Fabricators then process the resins into a

wide variety of products (Cohen et al. 2002). Roughly 99% of the industrial resins

produced from styrene can be grouped into six major categories. These six categories of

resins (including unsaturated polyester resins with and without reinforcement), and some

representative products made from the resins, are presented in Table 2-1.

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Table 2-1. Styrene use in industrial resin

Resin Type Estimated resin production (%)

Typical products produced from resins

Polystyrene 50 Construction materials, cups, plates, egg cartons, audio-visual equipment (e.g., cassettes), packaging, dairy containers, toys, furniture, industrial moldings (e.g., medical dental), insulation

Styrene-butadiene rubber 15 Tires, automobile parts (e.g., hoses, belts, seals, wire insulation)

Unsaturated polyester resins (glass reinforced)

12 Boats, tubs, shower stalls, spas, hot tubs, cultured marble products, building panels, trucks

Styrene-butadiene latexes 11 Backing for carpets and upholstery, paper coatings, floor tile, adhesives

Acrylonitrile-butadiene-styrene 10 Appliances, automobile parts, business equipment, construction materials, drains, ventilation pipes, hobby equipment, casings

Styrene-acrylonitrile 1 Appliances, automobile parts, housewares, battery casings, packaging

Unsaturated polyester resins (not reinforced)

Not reported Liners, seals, putty, adhesives

Source: Luderer et al. 2005.

The largest single use for styrene is in the manufacture of polystyrene (accounting for

roughly half of styrene use). Polystyrene is used extensively in the manufacture of plastic

packaging, thermal insulation in building construction and refrigeration equipment, and

disposable cups and containers. Styrene polymers and copolymers also are increasingly

used in the production of various housewares, including food containers, toys, and

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electrical devices, in the production of automobile body parts, corrosion-resistant tanks

and pipes, in various construction items, carpet backings, house paints, paper processing,

computer printer cartridges, insulation products, wood floor waxes and polishes,

adhesives, putties, personal care products, and other items (IARC 2002, Luderer et al.

2005, NLM 2008).

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Styrene is used as a cross-linking agent in polyester resins used in gel-coating and

laminating operations in the production of glass fiber–reinforced plastic products such as

boats, bathtubs, shower stalls, tanks, and drums (EPA 1997a, Miller et al. 1994). The

resins generally contain between 30% and 50% styrene by weight (EPA 1997a). Methyl

methacrylate may be used as a cross-linking agent instead of, or in addition to, styrene;

however, styrene is by far the most common agent used.

2.2 Production 12 There are two commercially viable methods to produce styrene (ATSDR 1992, HSDB

2008a). The most common process, which accounts for over 90% of the total world

styrene production, involves catalytic dehydrogenation of ethylbenzene. In the Dow

Process, superheated steam is injected with ethylbenzene over a fixed catalytic reactor.

The catalyst is iron-oxide based and contains Cr2O3 and KOH or K2CO3 as promoters

(Cheresources 2008b). Ethylbenzene conversion is typically 60% to 65%, and there are

three significant byproducts: toluene, benzene, and hydrogen. After the reaction, the

products are cooled and the product stream, which contains styrene, toluene, benzene,

and unreacted ethylbenzene, is fractionally condensed. After adding a polymerization

inhibitor, the styrene is vacuum distilled to reach the required purity (noted as 99.8%).

The second process involves oxidation of ethylbenzene to its peroxide, which is then

reacted with propylene to produce propylene oxide and alpha-methylphenyl carbinol. The

carbinol is then dehydrated to produce styrene.

As noted above, production of polystyrene is the single largest use of styrene. In one

process, an inert organic solvent environment provides the medium for the

polymerization reaction (Cheresources 2008a). 1,2-Dichloroethane is the most common

solvent used, although carbon tetrachloride, ethyl chloride, methylene dichloride,

benzene, toluene, ethylbenzene, and chlorobenzene are suitable. The preferred initiator


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for the reaction is a mixture of boron trifluoride and water. The typical feed stream to the

reactor consists of 50 weight percent styrene monomer, 100 ppm water and 200 ppm

boron trifluoride (based on styrene weight) with the remainder being organic solvent.

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The chemical reaction for synthesis of styrene from ethylbenzene and the polymerization

process for production of polystyrene, the most common product made from styrene, are

illustrated in Figure 2-1.

Figure 2-1. Synthesis of styrene from ethylbenzene and polymerization of styrene to form polystyrene

U.S. production of styrene has risen steadily since 1960 with a few dips from one year to

the next. In the Chemical Economics Handbook (CEH) Marketing Research Report for

styrene, Berthiaume and Ring (2006) estimated U.S. styrene production to be 1,740

million pounds in 1960, rising to a maximum of 11,897 million pounds in 2000, and

production of 11,387 million pounds in 2006. Figure 2-2 summarizes the historical

production data presented in CEH. Other sources, such as the U.S. International Trade

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Commission (USITC), have estimated similar production levels. In 2002, Cohen et al.

reported that U.S. styrene production exceeded 10 billion pounds [10,000 million pounds]

annually and that from this, over 13 billion pounds [13,000 million pounds] per year of

styrene-containing resins were produced.

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Figure 2-2. U.S. styrene production (1960–2006) Source: (Berthiaume and Ring 2006).

As of 2006 there were eight active producers of styrene in the United States. The three

largest of these producers accounted for 54% of domestic production in 2006

(Berthiaume and Ring 2006).

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Import and export data are presented in Figure 2-3. CEH (Berthiaume and Ring 2006)

and USITC data (USITC 2008a, 2008b) each showed a steadily increasing trend in both

imports and exports from 1975 through 2007. The minimum level for imports was 7

million pounds in both 1975 and 1977, and the maximum level was 1,475 million pounds

in 2007. The minimum level for exports was 574 million pounds in 1975 and the

maximum level was 4,200 million pounds in 2007.

During the 1990s, styrene consumption in the United States increased at an average

annual rate of 2.2% with over 99% consumed in the production of polymers and

copolymers (Berthiaume and Ring 2006). U.S. consumption of styrene in 2006 was 9,600

million pounds and was anticipated to reach a level of 10,800 million pounds by 2011.


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0500

10001500200025003000350040004500

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Figure 2-3. U.S. imports and exports for styrene Sources: Berthiaume and Ring 2006 and USITC 2008a,b.

2.3 Environmental release, fate, and occurrence 1 Styrene has been measured in both outdoor air and indoor air, with generally higher

levels found in indoor air. Styrene has been detected in a small percentage of U.S.

drinking water samples, generally at low levels, and it has also been detected in both

surface and ground waters in the United States. It has been found in soils of U.S.

hazardous waste sites. It can occur in food both naturally and through migration from

packaging materials containing residual styrene monomer. Numerous spills containing

styrene have been reported to the National Response Center (NRC) since 1990, and these

spills have the potential to contaminate air, water, soil, and even food supplies. This

section discusses the release, environmental fate, and occurrence of styrene in air, water,

soil, and food. The exposure level data presented in this section provides general

information on exposure levels and can be considered “semi-quantitative.” It is not

intended to provide an estimate of the level of exposure for the general population or for

any particular subpopulation. Section 5.2.1 discusses the possible estrogenicity of styrene

as an environmental contaminant.

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For environmental sampling, styrene is usually collected on solid sorbents (such as

charcoal), either directly for air samples or after purging in a gas stream for water, soil, or

solid waste samples (IARC 2002, ATSDR 1992). ATSDR noted that styrene from such


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samples can be measured very sensitively by capillary column gas chromatography with

flame ionization detector (GC/FID), and very specifically by gas chromatography with

mass spectrometric detection (GC/MS). ATSDR also noted that relatively low detection

limits and high accuracy can be achieved for the determination of styrene in

environmental samples. IARC reported that estimated detection limits for GC/FID

analysis of air samples ranged from 0.001 to 0.01 mg/sample. IARC also reported that the

practical quantitation limits are 5 μg/L for groundwater samples, 250 μg/L for water-

miscible liquid waste, 2,500 μg/L for non-water–miscible waste, 5 μg/kg for low-level

soil and sediment samples, and 625 μg/L for high-level soil and sludge samples.

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[While GC/FID and GC/MS are the most commonly used methods for the assessment of

styrene in environmental media, other methods are available and have been used in the

past. For data presented in this section and in the section on occupational exposure

(Section 2.5) the analytical methods often were not identified in the studies reviewed. It is

likely that different methods were used across these studies resulting in variations in the

quality of the data presented. The data that are presented span several decades, and

analytical methods are continually refined to obtain lower detection limits and to improve

accuracy and precision. Therefore, in some cases, the analytical methods used to obtain

these data may be outdated. There also will be differences in the quality of data presented

based on the purpose and strategy of the sample collection and the study design. The

environmental and occupational studies reviewed varied in the type of data provided. In

general, if arithmetic means and ranges were available, those data are presented here.

However, geometric means, medians, maximum values, standard deviations, and other

summary statistics may be provided if those data were presented rather than arithmetic

means and ranges in the cited document.]

The information reported for air, water, and soil is limited to data from the United States.

However, for styrene levels in food, information obtained from other countries are

provided in addition to U.S. data, as much of the food consumed in the U.S. is imported.


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2.3.1 Air 1 Styrene can be emitted in the air from industrial production and use of styrene and

styrene-based polymers and copolymers, motor vehicle emissions and other combustion

processes, off-gassing of building materials and consumer products, and cigarette

smoking (ATSDR 1992, IARC 1994a). For the general public, significant exposure to

styrene can result from both outdoor and indoor sources. The remainder of this section

discusses outdoor and indoor releases of styrene to air, its fate and transport in air, and

measured levels in outdoor and indoor air.

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2.3.1.1 Outdoor release 9 Major sources of styrene in outdoor air include industrial sources, automobile emissions,

and combustion processes such as waste incineration and the burning of wood (ATSDR

1992, IARC 1994a). Typical sources of industrial styrene emissions include facilities

producing styrene, polystyrene, other plastics, glass fiber–reinforced plastic products,

synthetic rubber, and resins (ATSDR 1992). For 2006 [the most recent data available],

the U.S. EPA’s Toxics Release Inventory (TRI) reported styrene fugitive air emissions of

9.9 million pounds, and point-source air emissions of 37.4 million pounds (TRI 2008a).

These air emissions combined [47.2 million pounds] accounted for roughly 93% of the

total TRI styrene releases for all reported environmental media in 2005. Between 1988

(the first year of TRI reporting) and 2005, the smallest reported total air release (point-

source plus fugitive emissions) was 30.3 million pounds in 1991 and the largest was 59.5

million pounds in 1999. Among the 519 TRI 2001 Core Chemicals, styrene had the 6th

highest level of point-source air emissions and the 5th highest level of fugitive air

emissions in 2005 (TRI 2008b, 2008c). [Note that since EPA’s reporting requirements are

for those facilities that produce or use large amounts of a chemical, actual emissions

probably are greater than those reported.]

Styrene has been identified in motor vehicle emissions from both gasoline- and diesel-

powered engines. The U.S. EPA estimated that in 1990, 32.9% of total U.S. styrene

emissions were from on-road vehicles (IARC 2002). In 1999, it was estimated that in the

U.S. 14,284 tons [28.6 million pounds] of styrene were emitted from highway vehicles

and 3,055 tons [6.1 million pounds] from non-road equipment (EPA 2007). Emissions in


9/29/08 17

2010 are expected to fall to 7,652 tons [15.3 million pounds] for highway vehicles and

2,297 tons [4.6 million pounds] for non-road equipment primarily as a result of

reductions due to the Mobile Source Air Toxics rule (see Section 2.7 for regulations and

guidelines).

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13

14

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28

Glass fiber–reinforced plastic composites production and boat manufacturing are other

major sources of styrene emissions. The U.S. EPA estimated that in 1990, 39.8% of U.S.

styrene emissions were from these sources (IARC 2002) (see Section 2.5.1 for further

discussion). [More recent data on national emissions levels from this industry were not

found.]

Another source of outdoor styrene emissions is thermal degradation of styrene-containing

polymers. IARC (2002) reported that results from one study showed styrene monomer to

be the main volatile product of the thermal decomposition of polystyrene, constituting up

to 100% of the volatiles. Styrene also has been measured in the air near open burning of

scrap tires. The EPA (1997b) reported a median concentration of 85 μg/m3 (20 ppb), with

a 90th percentile concentration of 2,320 μg/m3 (540 ppb) for ambient concentrations

measured within 1,000 feet downwind of 14 uncontrolled fires.

2.3.1.2 Indoor release 17 Indoor sources of styrene emissions include off-gassing of building materials and

consumer products and tobacco smoke (ATSDR 1992, IARC 2002). Styrene from

adhesives used in the construction and finishing of buildings has been identified in indoor

air. Polystyrene products such as packaging materials, toys, housewares, appliances,

computers, and other plastic and rubber items can also contribute small amounts of the

monomer to indoor air levels (ATSDR 1992, Bako-Biro et al. 2004).

EU (2002) noted that polystyrene and styrene copolymers are resistant to biodegradation,

and therefore, decomposition to the monomer is unlikely. However, polymers can contain

residual styrene monomer, and off-gassing of styrene from household products such as

carpet glues, construction adhesives, and polyester-containing flooring materials are

potential sources of styrene in indoor air (Luderer et al. 2005, EU 2002, ATSDR 1992).


18 9/29/08

Table 2-2 provides data on styrene monomer levels in various types of styrene polymer

and copolymer materials [more recent data were not found].

1

2

Table 2-2. Residual styrene-monomer levels in polymer and copolymer materials in 1980

Residual styrene levels (ppm)

Polymer or copolymer Typical Maximum Polystyrene 300–1,000 2,500 Acrylonitrile-butadiene-styrene (for food containers)

200–300 600

Acrylonitrile-butadiene-styrene (for other uses)

300–1,000 2,000

Styrene-acrylonitrile 600–1,200 2,000 Methyl methacrylate-butadiene-styrene ND–10 30 Glass-reinforced plastic 20–200 1,000 Styrene-acrylic copolymers 60 in latex NR Styrene-butadiene - raw polymer 10–30 NR Source: EU 2002. ND = not detected; NR = not reported.

Historical levels of styrene from 1976 to 1980 are presented in Table 2-3. These data

show some reduction in residual styrene monomer levels from 1976 to 1980. Residual

levels have been further reduced since this time due to improvements in production

methods (EU 2002). Luderer et al. (2005) noted that the residual monomer data from

1980 may overestimate residual levels of styrene in polymers currently manufactured in

the United States because of changes in regulations and production methods since 1980.

3

4

5

6

7

8


9/29/08 19

Table 2-3. Historical levels of residual styrene (mg/kg) in polymer and copolymer: 1976–1980

Polystyrene Expanded

polystyrene High-impact polystyrene

Acrylonitrile-butadiene-

styrene Styrene-

acrylonitrile

Year Mean Max Mean Max Mean Max Mean Max Mean Max 1976 870 970 – – 800 1,270 700b 1,600b – – 1977 700;

800a 1,020; 1,100a

– – 600 990 300b 1,060b 3,400 5,000

1978 380 580 1,400 – 420 840 300b 800b 1,000 1,550 1979 400 790 1,400 – 380 600 300b;

600c; 1,220d

700b; 790c; 1,220d

950 1,300

1980 410 600 1,000 – 360 490 300b; 600c; 700d

600b; 1,000c; 870d

950 1,250

Source: EU 2002. Max = maximum level reported. aTwo values were reported in original source without explanation. bIntended for food containers. cIntended for refrigerator applications. dIntended for household appliances.

There is limited information on losses of styrene to air from finished articles. EU (2002)

reported that based on an examination of three types of polystyrene to determine any loss

of residual monomer, no change was seen in flexible or rigid polystyrene cold drinks

cups over a six-month interval, although there appeared to be some loss (from 104 ppm to

71 ppm residual styrene) from foam hot drinks cups. Another study reviewed by the EU

reported that the residual monomer content of polystyrene and styrene copolymers (300

to 500 ppm) did not reveal any losses over 2 years. The EU also reported that typical

levels of styrene in expanded polystyrene molding will decrease from an initial level of

500 mg/kg to an equilibrium level of around 200 mg/kg over a period of 2 to 5 years

depending on use.

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3

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8

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12

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15

The European Union (EU 2002) reported that an emission factor of 0.03% [0.3 g/kg]

would be appropriate for long-term use (e.g., insulation in buildings) but would over-

estimate losses for short-term applications such as packaging. Rates of styrene emission

from glued carpet have been estimated at 98 ng/min per m2 (ATSDR 1992). In a chamber

test of cork parquet flooring applied on concrete, styrene emissions were measured at 3


20 9/29/08

μg/h-m2 after 24 hours, 2 μg/h-m2 after 168 hours, and < 1 μg/h-m2 after 576 hours [the

source of the styrene was not specified] (Uhde and Salthammer 2007).

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Infiltration of outdoor air has been proposed to be a potentially important source of

styrene levels in indoor air (Guo et al. 2004a). Infiltration of gasoline-related volatile

organic compounds (VOCs) from attached garages into houses is another potential source

of styrene in indoor air (Adgate et al. 2004b, Batterman et al. 2006, Batterman et al.

2007). In Minnesota residences, Adgate et al. found significantly higher levels of styrene

in homes with attached garages than in homes without attached garages. However, in

assessing styrene levels in houses and in garages of 15 Michigan residences with attached

garages, Batterman et al. found mixed results for styrene, with 8 of the 15 houses having

higher concentrations than the attached garages. The authors were unable to conclude that

styrene from the attached garages contributed to indoor air levels.

Increased styrene air concentrations have also been measured in the homes of smokers

versus non-smokers (IARC 2002, HSDB 2008a).

2.3.1.3 Fate 15 Styrene, with a vapor pressure of 6.4 mm Hg at 25°C, is expected to exist solely as a

vapor in the ambient atmosphere if released to air (HSDB 2008a). In its vapor phase, it is

expected to react rapidly with hydroxyl radicals and with ozone. Half-lives based on

these reactions have been estimated to range from 0.5 to 17 hours (Luderer et al. 2005).

Atmospheric washout is not expected to be an important process because of these rapid

reaction rates and styrene’s high Henry’s law constant.

Styrene levels in indoor air can be altered due to chemical reactions with other indoor

pollutants. Uhde and Salthammer (2007) reviewed how styrene and its aldehyde

degradation products fluctuated due to chemical surface interactions. They reported that

in a study that measured VOCs in a newly carpeted stainless-steel chamber in the

presence of ozone, the gas-phase concentrations of styrene, 4-vinyl-cyclohexene, and 4-

phenyl-cyclohexene decreased significantly while the concentrations of aldehydes

increased. The authors reported that both 4-vinyl-cyclohexene and 4-phenyl-cyclohexene


9/29/08 21

were suspected secondary pollutants from styrene-butadiene rubber, which was used for

foam backing for carpets.

1

2

4

5

6

7

8

9

10

11

12

13

14

2.3.1.4 Outdoor occurrence 3 The primary sources of styrene in outdoor air include emissions from industrial processes

involving styrene and its polymers and copolymers, vehicle emissions, and other

combustion processes (IARC 2002). ATSDR (1992) noted that styrene levels in outdoor

air are likely to be higher in urban areas than rural areas, and Alexander (1997) noted that

concentrations in outdoor air are generally higher in winter than summer months. The

Hazardous Substances Data Bank (HSDB 2008a) noted that except in highly polluted

areas, styrene concentrations in outdoor air generally are less than 1 μg/m3 [0.23 ppb];

although much higher levels have been reported. Table 2-4 summarizes reported

concentrations of styrene in outdoor air in the United States.

The U.S. EPA monitors ambient air concentrations of numerous air pollutants, including

styrene, throughout the United States, and these data are available on their AirData web

site (http://www.epa.gov/air/data/). Outdoor ambient air monitoring data for 259

monitoring sites were reported for 2007 (EPA 2008a). Based on 13,432 observations, the

mean concentrations for these sites ranged from 0.028 to 5.74 ppb and the maximum

concentrations ranged from 0.05 to 206.47 ppb. HSDB (2008a) and the European Union

(EU 2002) provided additional information on general or non-specific air concentrations

in the United States.

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22

23

24

25

26

27

28

29

IARC (2002), HSDB (2008a), European Union (2002), and Luderer et al. (2005) have

reported data on U.S. air levels of styrene in the vicinity of known sources (Table 2-4).

IARC (2002) reported ambient air levels of styrene in the vicinity of seven U.S.

reinforced-plastics processors. Higher levels were measured at distances of under 500 m

compared with levels at distances of 500 to 1,000 m.

High air levels have been reported in the vicinity of styrene-related industries, such as

reinforced plastic processors, or hazardous waste sites (see Table 2-4). Since the 1980s,

ATSDR has measured levels of various contaminants at hazardous waste sites during site

investigations, and summary data for these investigations are available through an online

http://www.epa.gov/air/data/


22 9/29/08

database called Hazardous Substance Release and Health Effects Database (HazDat)

(HazDat 2008). Between 1980 and 2005, outdoor air styrene concentrations measured on

the waste sites ranged up to 17,000 μg/m3 [4,000 ppb] and offsite concentrations ranged

up to 122 μg/m3 [28.6 ppb]. [Only maximum data are presented in the HazDat online

database.]

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3

4

5

6

7

8

9

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11

12

13

14

15

16

IARC (2002), Luderer et al. (2005), European Union (EU 2002), and HSDB (2008a)

have presented results of monitoring studies from U.S. urban areas (Table 2-4). Payne-

Sturges et al. (2004a) found that mean and median outdoor monitoring levels were less

than indoor air monitoring levels and personal monitoring levels (see “Indoor

Occurrence,” below); however, outdoor levels were higher than modeling results

predicted by the U.S. EPA’s Assessment System for Population Exposure Nationwide

(ASPEN) model. Similarly, Adgate et al. (2004a, 2004b) found higher styrene levels for

indoor air monitoring and personal monitoring than for outdoor ambient air monitoring.

For Table 2-4 and the remainder of the tables presenting environmental levels, the

number of samples is presented when it was available in the referenced source; otherwise,

the number of samples is not addressed.

Table 2-4. Concentrations of styrene in outdoor air in the United States

Location (year) Measurement Concentration

(ppb) Source General or unspecified locations Ambient air monitoring

throughout United States (2007)

mean levels maximum levels (based on 13,432 measurements)

0.028–5.74 0.05–206.47

EPA 2008a

California (1965) mean (range) 4.9 (1.9–14.8) EU 2002 Contra Costa County, CA (NR)

single measurement 0.09 HSDB 2008a

New Jersey, California residential areas

(NR)

range of medians of 6 sets of samples

0.07–1.0 EU 2002

Four unspecified states (1981–1984)

range ND–0.89 EU 2002

Unspecified locations (NR)

average of samples above detection limit from various studies (6,117 total samples)

0.14a HSDB 2008a


9/29/08 23

Location (year) Measurement Concentration

(ppb) Source Unspecified location(s) (NR)

median concentration from 135 samples

2.1 HSDB 2008a

Measurements in the vicinity of a known source Vicinity of reinforced-plastics

processors in multiple states

(NR)

< 500 m from facility 500 to 1,000 m from facility

0.07–690 0.4–5.6

HSDB 2008a, IARC 2002

Houston, TX, industrial complex close to major transport route

(1987–1988)

mean of 135 samples 0.5a EU 2002

Vicinity of sanitary and hazardous waste landfills (NR)

maximum range of means

15.5 0.12–1.53

HSDB 2008a

Hazardous waste sites (1980–2005)

onsite measurements offsite measurements

up to 4,000 up to 28.6

HazDat 2008

Allegheny mountain tunnel, Pennsylvania (NR)

range 0.3–1.6 HSDB 2008a

Caldecott Tunnel, San Fransciso, CA (NR)

range 9.83–36.73 HSDB 2008a

Pennsylvania turnpike tunnel (NR)

range 0.25–1.5 Luderer et al. 2005

Urban locations Baltimore, MD (2000–2001) mean

median 0.12a 0.06a

Payne-Sturges et al. 2004a

Minneapolis, MN (1997) median, winter median, spring

0.023a 0.0

Adgate et al. 2004a

Minneapolis, MN (2000) mean 0.12a Adgate et al. 2004b

Phoenix, AZ (1994–1996) range of means 0.49–5.64 HSDB 2008a Tucson, AZ (1994–1996) range of means 0.09–0.23 HSDB 2008a Los Angeles, CA (1981) range; 16 of 17 samples

positive 0.5–3.0a EU 2002

Three New Jersey cities (NR) range of means, summer range of means, winter

0.07–0.13 0.15–0.23

Luderer et al. 2005

Twenty urban test stations in California (1989–1995)

1 d per month monitoring average maximum

0.2 2.9

IARC 2002

Four unspecified cities (NR) range 1–15 HSDB 2008a One unspecified city (NR) median 0.14 HSDB 2008a ND = not detected; NR = not reported. a Reported in units of μg/m3 in source document.


24 9/29/08

2.3.1.5 Indoor occurrence 1 Indoor air concentrations of styrene typically exceed outdoor air concentrations (Miller et

al. 1994). IARC (2002) reported that median residential air concentrations collected by

personal sampling have generally ranged from 1 to 3 μg/m3 [0.2 to 0.7 ppb]. ATSDR

(1992) noted that mean indoor air levels of styrene have been reported in the range of 0.1

to 9 μg/m3 [0.02 to 2.1 ppb] and can be attributed to off-gassing from building materials

and consumer products and from tobacco smoke. Fishbein (1992) reported typical indoor

levels ranging from 0.3 to 50 μg/m3 [0.07 to 11.7 ppb]. Based on a U.S. EPA national

VOCs database compiled in the early- to mid-1980s from various sources of U.S. indoor

air concentration data, Miller et al. (1994) reported a mean indoor styrene air level of

1.413 ppb and a median level of 0.305 ppb based on 2,125 data points. Styrene levels

measured in indoor air in the United States are presented in Table 2-5.

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6

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10

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12

13

14

15

16

17

18

19

20

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24

25

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27

28

29

30

Increased styrene air concentrations have been measured in the homes of smokers versus

non-smokers. In a screening-phase study of 284 Minnesota homes, Adgate et al. (2004b)

found statistically significant increases in styrene levels in homes with smokers compared

with homes without smokers. Another study showed that styrene concentrations were

approximately 0.5 μg/m3 [0.1 ppb] higher in homes with smokers than in homes without

smokers (IARC 2002). Tobacco use by adults also resulted in elevated styrene exposure

levels for children (Adgate et al. 2004a, Adgate et al. 2004b) (see below). Based on a

styrene emission factor of 235 μg/cigarette, Nazaroff and Singer (2004) estimated

exposure concentrations ranging from 0.6 to 1.4 μg/m3 [0.14 to 0.33 ppb] in U.S. private

residences.

Payne-Sturges et al. (2004a) noted that exposure research has consistently shown

personal exposure levels for most VOCs are very different from outdoor air

concentrations and this may result in over- or under-estimates of risks when outdoor air

concentrations are used exclusively. The authors examined the extent of exposure

misclassification and its effect on risk as estimated by the U.S. EPA’s ASPEN model

relative to monitoring results from a community-based exposure assessment conducted in

Baltimore, MD. For styrene, monitoring data were consistently higher than the levels

predicted by the ASPEN model. The ASPEN model predicted mean and median air


9/29/08 25

concentrations for styrene of 0.12 μg/m3 [0.03 ppb]; however, mean monitoring values

were 2.72 μg/m3 [0.64 ppb] for indoor air, and 2.51 μg/m3 [0.59 ppb] for personal

monitoring. The authors noted that indoor exposures were the dominant source of styrene

exposure.

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5

6

7

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11

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13

14

15

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19

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In a study of exposure to VOCs in microenvironments, breathing zone styrene air

concentrations were measured for a non-random sample of 71 non-smoking adults living

in three urban neighborhoods in Minneapolis-St. Paul, MN and compared with

concurrent area measurements taken inside the participants’ residences and in outside air

(Sexton et al. 2007). The participants maintained time-activity logs during the sampling

period, recording the amount of time spent in seven microenvironments: (1) indoors at

home, (2) indoors at work or school, (3) indoors in other locations (any indoor location

other than home, work, or school); (4) outdoors at home, (5) outdoors at work or school,

(6) outdoors in other locations (any outdoor location other than home, work, or school);

and (7) in transit. The authors reported that the highest estimated concentrations

(presented graphically only) were found for “indoors in other locations” followed by

“indoors at work/school” and “indoors at home.” The means for “outside” and “in transit”

were only slightly greater than zero. Batterman et al. (2002) also reported low levels of

styrene in a study of VOCs in microenvironments related to transportation (buses and

cars), and suggested that industrial emissions contributed to variation in the levels

measured.

Loh et al. (2006) characterized the distribution of VOCs, including styrene, in non-

residential microenvironments in stores, restaurants, and transportation modes in the

Boston, MA metropolitan area as part of the Boston Exposure Assessment in

Microenvironments (BEAM) study. They reported that styrene levels were higher in

stores, particularly hardware, housewares, and multipurpose stores, compared with

transportation. Styrene varied significantly (P < 0.05; Wilcoxon rank sum test) by season,

with levels being higher in summer compared with winter; however, only hardware and

multipurpose stores were sampled in both seasons. The authors also concluded that

concentrations of styrene were strongly influenced by smoking in the dining


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microenvironment. Uhde and Salthammer (2007) noted that numerous chemical

interactions can impact indoor air levels of styrene.

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8

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12

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15

In a study assessing VOC exposures to children in Minneapolis, MN, Adgate et al.

(2004a) noted that styrene levels were more frequently detectable in home and personal

samples than in outdoor samples or samples taken in the children’s schools. The authors

reported slightly higher styrene air levels for home monitoring than for personal

monitoring, and an 8-fold to almost 10-fold increase in home levels compared with

school levels. In another study of VOC exposures in households with children (N = 284)

in Minneapolis, MN, households with smokers, households with attached garages, and

non-urban residences (which had a greater prevalence of smokers and attached garages)

all had significantly higher levels of styrene (Adgate et al. 2004b).

ATSDR measures indoor air concentrations as part of their hazardous waste site

investigations (HazDat 2008). Between 1980 and 2005, maximum concentrations

measured in buildings onsite at hazardous waste sites were much higher than maximum

concentrations measured in off-site buildings.

Table 2-5. U.S. levels of styrene measured in indoor air and by personal monitoring Location (year) Measurement Concentration (ppb) Source General data Nationwide (compiled from data before mid-1980s)

mean median

1.413 0.305

Miller et al. 1994

Studies assessing microenvironments Minneapolis, MN (2000)

School monitoring winter (median) spring (median)

Home monitoring winter (median) spring (median)

Personal monitoring winter (median) spring (median)

0.02a (N = 39) 0.02a (N = 47)

0.16a (N = 93) 0.19a (N = 88)

0.12a (N = 93) 0.12a (N = 88)

Adgate et al. 2004a

Minneapolis, MN (1997)

Screening assessment indoor monitoring (mean)

Intensive-phase assessment personal monitoring (mean)

0.28a (N = 284)

0.28a (N = 73)

Adgate et al. 2004b


9/29/08 27

Location (year) Measurement Concentration (ppb) Source indoor monitoring (mean) 0.33a (N = 101)

Baltimore, MD (2000–2001)

indoor monitoring (mean) personal monitoring (mean)

0.64a (N = 33) 0.59a (N = 31)

Payne-Sturges et al. 2004a

Minneapolis-St. Paul, MN (1999)

home (mean) school/work (mean) other indoor (stores, restaurants) (mean)

outside (mean) in transit (mean) (N = 333 personal exposure samples (including duplicates) with matched time activity data for 70 participants)

0.14a,b 0.19a,b 0.26a,b

< 0.01a,b < 0.01a,b

Sexton et al. 2007

Detroit, MI commuting routes (1999)

Pilot study range Three day study mean and range

0.07–0.26a (N = 16)

0.26a (0.02–0.82) (N = 48)

Batterman et al. 2002

Boston, MA (2003–2004)

stores (geometric mean) restaurants (geometric mean)

0.71a (N = 89) 0.28a (N = 20)

Loh et al. 2006

Hazardous waste sites Hazardous waste sites (1980–2005)

On-site buildings (maximum) Off-site buildings (maximum)

1,276a 41.5a

HazDat 2008

a Reported in units of μg/m3 in source document. b Estimated from graph.

2.3.2 Water 1 2.3.2.1 Release 2 The primary source of styrene in surface waters is industrial discharges (ATSDR 1992).

Styrene has been detected in effluents from chemical, textile, latex, and coal-gasification

plants at levels up to 970 μg/L. The daily styrene loading from a single chemical plant

into the St. Clair River (Michigan) was estimated at 133 kg [293 lb]. Styrene also has

been detected in the leachate from an industrial landfill and in surface water and

groundwater at U.S. hazardous waste sites.

3

4

5

6

7

8

9

10

11

12

For the year 2006, a reported 4,043 lb of styrene were released to U.S. surface waters

based on TRI data (TRI 2008a). The TRI data have fluctuated widely since 1988, with a

maximum release of 243,148 lb reported in 1998 and a minimum of 3,004 lb in 2001.

The second-highest reported release to surface water was 59,069 lb in 1988. Styrene also


28 9/29/08

has been detected in both the groundwater and surface water at hazardous waste sites

(ATSDR 1992).

1

2

4

5

6

7

8

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2.3.2.2 Fate 3 Volatilization and biodegradation are expected to be the major fate and transformation

processes in water. Based on its Henry’s law constant, styrene is expected to volatilize

rapidly from environmental waters: the extent of volatilization depends on water depth

and turbulence with no volatilization occurring in stagnant deep water (ATSDR 1992,

Luderer et al. 2005). The estimated volatilization half-life of styrene from a river 1 m

deep with a current speed of 1 m/s and a wind velocity of 3 m/s is roughly 3 hours. Half-

lives have been estimated from 1 hour in a shallow body of water to 13 days in a lake,

and from 4 to 30 weeks in groundwater (Luderer et al. 2005). Some biological oxygen

demand studies have shown styrene to be biodegradable. Hydrolysis is not expected to be

an important degradation process. Adsorption to particulate matter and sediment may

have some significance, based on an organic carbon adsorption coefficient (Koc) of 270 to

550 (Howard 1989). Styrene generally does not persist in water, because of its

biodegradability and volatility (Cohen et al. 2002).

2.3.2.3 Occurrence 17 Limited data are available on styrene levels in water. When styrene has been detected in

waters, it has generally been at low levels. This section discusses styrene levels in

drinking water and environmental waters, and levels that have been detected in various

waters at hazardous waste sites. Table 2-6 presents monitoring results for styrene in U.S.

waters.

Drinking water 23 Extensive studies of U.S. drinking-water supplies indicate that if styrene is present, it

generally is at very low concentrations (< 1 μg/L [1 ppb]) (Cohen et al. 2002). Miller et

al. (1994) reported that in surveys of drinking-water supplies in the United States and

Canada, styrene has been detected in a small percentage of drinking-water samples at

concentrations generally less than 1 μg/L [1 ppb]. Styrene was not detected in several

U.S. drinking-water surveys (Miller et al. 1994, EU 2002); detected but not quantified in

other studies (Howard 1989); and reported as detected in some studies styrene, but no


9/29/08 29

levels were reported (Miller et al. 1994, Howard 1989). Levels have been reported in

drinking-water supplies in Cincinnati, OH, in Iowa well water, and in Connecticut in well

water adjacent to a landfill that contained styrene buried in drums (Howard 1989).

1

2

3

5

6

7

9

10

11

12

13

14

Environmental water 4 Styrene has been found in the lower Tennessee River, the Kanawha River in West

Virginia, the Great Lakes, and detected but not quantified in the Delaware River (EU

2002, Howard 1989)

Hazardous waste sites 8 In the ATSDR Toxicological Profile for Styrene, it was reported that the geometric mean

levels of styrene at U.S. hazardous waste sites were 9.3 μg/L [9.3 ppb] for surface water

and 5.3 μg/L [5.3 ppb] for groundwater (ATSDR 1992). Styrene has been measured as

part of ATSDR’s hazardous waste site investigations, which includes monitoring of on-

site and off-site groundwater, on-site and off-site surface waters, and on-site tap water

(HazDat 2008).

Table 2-6. Levels of styrene measured in U.S. waters Water type, location (year) Additional information

Concentration (ppb) Source

Drinking water Drinking water, unspecified location (1975–1981)

3 surveys and over 1,000 samples

ND Miller et al. 1994

Drinking water, Cincinnati, OH (NR)

no additional information provided

0.024 Howard 1989

Drinking water, unspecified location (1977–1981)

102 surface water sources 12 groundwater sources

ND ND

EU 2002

Drinking water, KS, MO, and NB (1982)

drinking water collected from 272 sites


Drinking water, Evansville, IN (NR)


NQ Howard 1989

Drinking water, Cleveland, OH (NR)


NQ Howard 1989

Drinking water, New Orleans, LA (NR)

contamination might have come from the filter

NR Howard 1989

Well water, IA (NR) no additional information provided

1.0 Howard 1989


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Water type, location (year) Additional information

Concentration (ppb) Source

Well water, CT (1962) well water adjacent to a landfill containing styrene buried in drums

100–200 Howard 1989

Well water, WI (early 1980s)

detected in only 1 of 1,791 private and community wells

NR Miller et al. 1994

Groundwater, unspecified locations (NR)

945 groundwater samples in a drinking-water survey


Environmental water Surface water, lower Tennessee River (NR)

single water sample 4.2 Howard 1989

Surface water, Kanawha River, WV (NR)


1.0 Howard 1989

Great Lakes (1982–1983)

winter (average) spring (average) summer (average)

0.2 0.5

< 0.1

EU 2002

Delaware River (NR) no additional information provided

NQ Howard 1989

Hazardous waste sites Tap water, nationwide (1980–2005)

onsite tap water (one measurement)

0.8 HazDat 2008

Surface water, nationwide (NR)

geometric mean level at hazardous waste sites

9.3 ATSDR 1992

Surface water, nationwide (1980–2005)

onsite levels (maximum) offsite levels (maximum)

26,000 0.4

HazDat 2008

Groundwater, nationwide (NR)

geometric mean level at sites 5.3 ATSDR 1992

Groundwater, nationwide (1980–2005)

onsite groundwater monitoring wells (maximum)

offsite groundwater monitoring wells (maximum)

onsite private wells (maximum) offsite private wells (maximum) municipal groundwater well near a hazardous waste site (maximum)

55,000

40,000

5,000 30 1.6

HazDat 2008

ND = not detected, NQ = detected but not quantified, NR = level not reported.

2.3.3 Soil 1 2.3.3.1 Release 2 Soil may become contaminated through spills or discharges of styrene-containing

materials and through land disposal of styrene-containing wastes (ATSDR 1992).

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Sediment may become contaminated through disposal of styrene-containing wastes to

surface waters or through overland transport of contaminated materials to surface waters.

For 2006, TRI data showed that 11,242 lb of styrene were released to land (on-site and

off-site land treatment and other land disposal) (TRI 2008a).

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2.3.3.2 Fate 5 Styrene in soils is subject to biodegradation. Degradation of 87% to 95% has been

observed in sandy loam and landfill soil over a 16-week period, and degradation of 2.3%

to 12% per week has been observed in two subsurface aquifers (Howard 1989). Koc

values ranging from 260 to 550 have been calculated (Howard 1989, Luderer et al. 2005).

These Koc values indicate moderate to low soil mobility. It has been demonstrated that

styrene buried in soil can leach into underlying groundwater. Styrene that leaked into

surrounding soil from buried drums persisted for up to two years. Relatively strong

adsorption of styrene was observed in a sand aquifer, as the breakthrough time for styrene

was about 80 times that of a nonadsorbing tracer (Howard 1989). Varying rates of

volatilization from soils have been reported in the literature; however, all studies agree

that volatilization rates decrease with increasing soil depth (Luderer et al. 2005).

2.3.3.3 Occurrence 17 There are limited data on styrene levels in soil. ATSDR has measured sediment and soil

concentrations of styrene as part of numerous hazardous waste-site investigations

(HazDat 2008). The maximum concentrations measured in sediment were 70 ppm on-site

and 0.37 ppm off-site. Soil concentrations were obtained at differing soil depths both on-

site and off-site. For samples taken from the top three inches of soil, concentrations on-

site were up to 14,000 ppm and off-site concentrations were up to 0.14 ppm. Surface top-

soil concentrations on-site were measured at levels up to 2,900 ppm. Subsurface soil

(deeper than 3 inches) was only measured on-site with a maximum concentration of

4,600 ppm. Because styrene is volatile, it is also present in soil gas and was measured

during the waste-site investigations both on-site and off-site. On-site soil gas

concentrations were up to 8,082,000 μg/m3 [1,896 ppm], and off-site concentrations were

up to 690 ppb [0.69 ppm]. Sediment from the lower Tennessee River contained styrene at

4.2 ppb [0.0042 ppm] (Howard 1989).


32 9/29/08

2.3.4 Food 1 2.3.4.1 Sources of styrene in food 2 Styrene has been detected as a constituent of a wide range of foods and beverages, with

the highest measured levels occurring in unprocessed, raw cinnamon (IARC 1994a).

Styrene is known to occur in the exudates from damaged trunks of certain trees, probably

from the natural degradation of the cinnamic acid derivatives that occur in large

quantities in the exudates, and this has been proposed as the source of styrene in

cinnamon (IARC 1994a). Pinches and Apps (2007) demonstrated that in the presence of

cinnamic acid, the molds Trichoderma viride and T. koningii produced styrene in foods.

Styrene is also known to occur at very low concentrations in many agricultural foods,

although it is not known whether the styrene is produced endogenously or is the result of

environmental contamination (Tang et al. 2000). The presence of styrene in packaged

foods is reported to be due primarily to monomer leaching from polystyrene containers

(ATSDR 1992, Howard 1989). The primary factors that determine the rate of migration

of styrene from polystyrene containers include the lipophilicity of the food, surface area

of the container per volume of food, and the duration of contact (ATSDR 1992, EU 2002,

Lickly et al. 1995a).

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ATSDR (1992) reported that the rate at which styrene migrates from polystyrene

containers into food is mainly a function of the diffusion coefficient of the monomer in

the polymer and of the lipophilicity of the food. For example, 4% to 6% of the free

monomer in polystyrene packaging migrated into corn oil or sunflower oil within 10

days, while only 0.3% to 0.6% migrated into milk, beef, or water. Stoffers et al. (2004)

found the mean styrene migration rate from polystyrene into olive oil stored at 40°C for

10 days to be 0.013 mg/dm2 [130 ng/cm2]. The authors noted that the migration was quite

low: only 1.8% of the initial styrene migrated. ATSDR (1992) reported styrene migration

from foam cups into liquids such as water, tea, or coffee to be about 8 ng/cm2, while

migration into 8% ethanol, as might be encountered in wine or other alcoholic drinks,

was 36 ng/cm2.

Lickly et al. (1995a) found that styrene migration from polystyrene foam used for food-

contact materials (styrofoam plates, bowls, cups, egg cartons, meat trays, and hinged


9/29/08 33

carryout containers) was proportional to the square root of the time of exposure. Others

have noted that styrene concentrations increase in foods packaged in polystyrene with

increasing duration of contact (ATSDR 1992, Lozano et al. 2007, Miller et al. 1994).

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Temperature also has an impact on styrene migration. Lickly et al. (1995a) reported that

the log of the mean diffusion coefficient was linearly related to the inverse of the absolute

temperature of exposure from 70°F to 150°F [21°C to 66°C]. The mean diffusion

coefficients ranged from 4.5 × 10-11 cm2/s at 70°F to 3.4 × 10-9 cm2/s at 150°F. Choi et al.

(2005) examined the migration behavior of styrene monomer and oligomers from

polystyrene to the food simulants water and heptane, which are used to simulate aqueous

and fatty foods, respectively. Higher temperatures yielded faster migration rates, and the

higher molecular weight oligomers had slower migration rates than the styrene monomer.

Styrene can migrate into food from plastic containers during heating or cooking in

microwave or conventional ovens. Nerín and Acosta (2002) estimated the migration of

styrene and several other VOCs into food from five commercially available types of

plastic containers: polycarbonate, polypropylene copolymer, polypropylene random,

polypropylene-20% talcum, and styrene-acrylonitrile. Styrene migration was estimated at

levels ranging from 1.8 × 10-6 to 6.7 × 10-4 mg/kg of food. The experiment was conducted

in 120°C to 150°C [250°F to 300°F] ovens for 30 minutes. The maximum migration level

was from a styrene-acrylonitrile container, and the minimum migration level was from a

container made of polypropylene copolymer.

In an assessment of the effects of cold storage and packaging material on the migration of

a number of chemicals, including styrene, into sweet-cream butter, Lozano et al. (2007)

found that the relative abundance of styrene in foods increased as a function of time and

storage temperatures. Styrene levels were found to be lower for fresh and frozen butter

products when compared with refrigerated butter products (see Table 2-7). Styrene levels

were also found to be higher for butter products wrapped in parchment when compared

with butter wrapped in foil.

Styrene has a log Kow of 2.95, indicating moderate potential for bioaccumulation

(Howard 1989). Howard (1989) suggested that styrene’s solubility (“relatively high water


34 9/29/08

solubility”) to be high enough to make bioconcentration in biological organisms unlikely.

Based on a bioconcentration factor (BCF) of 13.5, bioconcentration of styrene in aquatic

organisms is expected to be low (HSDB 2008a). EU (2002) did an extensive review and

analysis of the BCF and similarly concluded that it is unlikely that styrene will

accumulate in aquatic organisms. However, styrene has been detected in fish and other

aquatic organisms (see Section 2.3.4.2 below).

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2.3.4.2 Styrene levels in food 7 Styrene levels in foods have been extensively documented. As noted above, styrene can

leach from containers and wrapping materials into food, and it also can occur naturally in

foods. This section first presents data on styrene levels in food due to migration from

packaging materials. This is followed by a discussion of levels in food not believed to be

due to migration from packaging materials, or the source of styrene is not known. Lastly,

data are provided for the U.S. FDA’s Total Diet Study, which simply measures levels of

contaminants, including styrene, in table-ready food, irrespective of the source of the

contaminant.

Miller et al. (1994) and HSDB (2008a) summarized the results of several studies that

measured styrene concentrations in foods packaged in polystyrene. Table 2-7 summarizes

these data.

Table 2-7. Measurements of styrene in foods packaged in polystyrene

Food Concentration, mean or rangea

(μg/kg, or ppb) Dairy Products Butter (range from fresh to 12 months of storage)

Wrapped in parchment Wrapped in foil Refrigerator-stored after 12 months Freezer-stored after 12 months

22.7–1,174 0–277

277–1,174 101–607

Sour cream 143–246 Yogurt trace–34.6 Butter-fat cream 59.2 Milk 17.2 Soft cheese 16


9/29/08 35


(μg/kg, or ppb) Cream 11 Margarine table spreads 10 Cottage cheese 9.3 Beverages Orange drink 47 Lime drink 25 White coffee 21 Cold lemon drink 17 Hot chocolate 13 Desserts Cream dessert products 30 Other unspecified desserts 22 Fruit Glacé fruit < 10 Strawberries < 10 Other Products Chopped peel (unspecified fruit) 180 Gravy 64 Honey 22.7 Coleslaw < 10 Fish < 10 Fresh meat < 10 Takeout food < 10 Eggs ND Wine ND Sources: HSDB 2008a, Lozano et al. 2007, Miller et al. 1994. ND = not detected (levels of detection not provided). aA range is provided if the source document provided a range or if data are combined across sources.

Based on a literature review, Cohen et al. (2002) presented data on styrene levels

measured in various foods (Table 2-8). Styrene occurs naturally in some foods and

beverages (Miller et al. 1994, Steele et al. 1994, Tang et al. 2000), [and although it was

not specified whether levels presented by Cohen et al. were measured by a process that

avoided contact with styrene, it is likely that these data represent naturally occurring food

levels].

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Table 2-8. Food levels of styrene [source of styrene unknown]


(μg/kg, or ppb) Fruits Black currants 60 Bilberries 25 Kiwi 2 Soursop 0.17 Papaya 0.1 Sapodilla fruit 0.01 Vegetables Peas, southern 0–20 Lentils 5 Peas, split 5 Beans 4 Fish Whitefish 1 Meat Turkey sausage 100 Guinea hen, roasted (in skin) 1 Eggs 1–6 Alcoholic beverages Beer 10–200 Red wine 0–10 Bilberry wine < 10 Hot beverages Roasted coffee 20–360 Source: Cohen et al. 2002. a A range is provided if data were provided as a range or as multiple entries in the source.

Using a process that avoided contact with styrene or any type of plastic, Steele et al.

measured styrene concentrations in 12 types of raw agricultural products, with results

suggesting that styrene may be a natural constituent of many foods. Of 12 foods, 8 had

detectable styrene levels, from a low of 0.233 ng/g [ppb] for Oregon peaches to a high of

39,200 ng/g [ppb] for cinnamon from Indonesia. [It is noteworthy that three cinnamon

samples from three different sources were analyzed, and concentrations ranged from 179

ng/g to the high of 39,200 ng/g.] Other studies that have measured natural levels in foods

(i.e., without contact with polystyrene) have yielded similar results (Miller et al. 1994).

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Styrene also is produced naturally as a metabolite in the process of making some foods,

such as wine, beer, and cheese (Cohen et al. 2002); it has been measured in wines, with

the majority of samples showing concentrations of 1 to 3 μg/L; the maximum

concentration observed was 8 μg/L (Tang et al. 2000).

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Whole body concentrations of styrene ranging between 15 and 100 μg/kg have been

measured in splake and walleye fish caught in the St. Clair River, Canada. Styrene was

also detected, but not quantified, in several other fish from the St. Clair River (EU 2002).

Edible shellfish from Atlantic Canada were reported to contain styrene at levels less than

10.0 μg/kg. In a Japanese survey in 1986, styrene was found in 28 of 131 samples of fish

at concentrations ranging from 0.5 to 2.3 μg/kg (limit of detection 0.5 μg/kg).

Since 1991, the U.S. FDA has measured styrene in U.S. foods in its Total Diet Study

(TDS). The TDS measures levels of various contaminants and nutrients in foods that are

prepared as they would be consumed, so the results can be used to provide a realistic

measure of intake. Foods are purchased from supermarkets in selected U.S. cities,

generally three to four times per year, and shipped to a central FDA laboratory, where

they are prepared and analyzed (foods are measured as raw commodities if they are

generally consumed as such). Table 2-9 summarizes styrene levels detected in TDS food

samples from 1991 through 2003 (the most recent year for which data were available).


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Table 2-9. Summary of styrene levels in FDA’s Total Diet Study (1991–2003a) Food group (number detected/number of samples) Range of

meansb (ppb)c

Full range across samples

(ppb)c Fruits and vegetables (raw oranges, bananas, avocados, strawberries, tomatoes, raisins; frozen strawberries; canned corn), and fruit juices (57/359)

0.1–119.26 < 2.0–1,980

Breads (white bread, fruit or plain muffins) (38/88) 5.23–29.07 < 2.0–510 Desserts and sweets (ice cream, cookies, cakes, Danish pastry, fruit pies, candy, brownies, chocolate, popsicle, doughnut, toaster pastry, soda pop, sandwich cookies) (287/792)

0.05–50.77 < 2.0–199

Dairy (cheese, cream cheese, butter, milk, sour cream) (51/308) 0.05–11.11 < 2.0–196 Snacks (roasted nuts and sunflower seeds, peanut butter, oil-popped and microwave popcorn, tortilla and potato chips, crackers) (134/352)

0.25–37.65 < 2.0–116

Fast food and takeout (hamburger, hotdog, pizza, taco, beef chow mein, French fries, fried chicken) (170/484) 0.28–17.95 < 2.0–94

Meat, fish, eggs (cooked ground beef, chuck roast, pork sausage and bacon, lamb, turkey, bologna, frankfurter, salami, tuna, fish sticks, scrambled eggs) (152/572)

0.23–7.59 < 2.0–85

Infant products (soy-based and milk-based formula, teething biscuits, apple juice, carrots, beef and broth/gravy) (8/264) 0.05–1.82 < 2.0–80

Oil products (olive, safflower, and vegetable oil; margarine) (45/92) 1.25–46.5 < 2.0–115

Breakfast cereals (fruit flavored, granola with raisins) (10/88) 0.48–1.77 < 2.0–50 Salads (macaroni and potato salad, coleslaw, buttermilk-type salad dressing) (11/56) 0.25–4.5 < 2.0–8.0

Source: FDA 2006. a The most recent year for which data were available as of April 2008. b In calculating the means, FDA assigned a level of 0 to results below the limit of detection. c Data presented in ppm in source document.

2.4 General population exposure 1 This section provides information related to exposure to styrene for the general

population. Because most exposure estimates are not specific to a particular country, and

international data often are utilized in the assessments, some exposure estimates are

presented that are not specific to the United States. This information may be useful in

identifying the factors that impact exposure for the general population in the United

States and elsewhere.

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Sources of exposure to styrene include inhalation (including indoor and outdoor ambient

air, smoking, and inhalation of environmental tobacco smoke), dermal exposure, and

consumption of contaminated food, water, and other beverages. Increased exposures


9/29/08 39

could occur for persons living in urban areas or close to major sources of styrene (e.g.,

highly trafficked areas, industrial production facilities, or hazardous waste sites). Another

potential source of exposure to the general public is exposures from inadvertent chemical

spills (NRC 2008).

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Exposure from ingestion of municipal drinking-water supplies probably is negligible, as

styrene has been detected in drinking-water monitoring surveys infrequently, and when it

has been detected, it has generally been at low levels. Ingestion of contaminated

groundwater, however, could result in significant exposure (ATSDR 1992, Howard

1989). Because of the low occurrence and levels of styrene in water, Cohen et al. (2002)

suggested that dermal exposures from water could be assumed to be negligible.

Fishbein (1992) estimated the relative significance of different routes of exposure to

styrene to illustrate the importance of both indoor air exposures and occupational

exposures. The results of this analysis are presented in Table 2-10. These results show

that occupational exposures result in the highest styrene intakes; however, the general

public also is exposed to styrene.

Table 2-10. Daily styrene intakes for the general public from various sources

Exposure situation Styrene concentration

(ppb)a

Nominal daily intake

(μg)

Daily intake for 70 kg adult

(μg/kg bw/day) Within 1 km of the production unitb

7.0 600 9

Polluted urban atmosphereb 4.7 400 6 Urban atmosphereb 0.07 6 0.09 Indoor airb 0.07–11.7 6–1,000 0.09–14 Polluted drinking water (2 L per day)

0.2 2 0.03

Cigarette smoke (20 cigarettes per day)

4.7–11.3 400–960 6–14

Source: Fishbein 1992, Luderer et al. 2005. a Presented in units of μg/m3 in source document. b Based on the assumption of a daily breathing volume of 10 m3 at work or 20 m3 at home or in an urban environment.

Health Canada estimated daily styrene intakes from various media for different age

groups of the Canadian general population (Table 2-11). As seen in Table 2-11, food and

indoor air are the largest contributors to exposure for non-smokers. In this assessment, an

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indoor air concentration of 0.28 μg/m3 was used, which is similar to the low-end value

used by Fishbein (1992) (above). Estimates for exposure from smoking assumed that

styrene content in mainstream cigarette smoke is 10 μg/cigarette [which was half the

level of the minimum of the range of values presented by Fishbein above], and that 20

cigarettes per day are smoked. [Note that the exposure values in this table are presented

in units of μg/kg b.w. and are not directly comparable to most of the data in this section,

which are presented in units of μg/d.]

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Table 2-11. Estimated daily intake of styrene from various media for Canadians of different ages

Estimated intake (μg/kg b.w./day)

Medium 0–6 mo 7 mo–4 yr 5–11 yr 12–19 yr 20–70 yr Air ambient 0.004– 0.11 0.006 – 0.15 0.007–0.17 0.006–0.14 0.005–0.13 Indoor 0.07 0.09 0.10 0.09 0.08 Drinking water < 0.005–0.03 < 0.00 –0.02 < 0.002–0.08 < 0.001 – 0.006 < 0.001–0.005 Soil < 0.00005 < 0.0004 < 0.00001 < 0.000004 < 0.00003 Food < 0.58 < 0.53 < 0.30 < 0.15 < 0.11 Total intake (not including cigarettes)

< 0.66– < 0.79 < 0.63– < 0.79 < 0.41– < 0.58 < 0.25– < 0.39 < 0.20– < 0.33

Intake by cigarette smokers

NA NA NA 3.51 2.86

Source: HealthCanada 1993. NA = not assessed.

Smoking can result in styrene exposure both directly for smokers and indirectly through

environmental tobacco smoke (ETS) (i.e., side-stream smoke and exhaled cigarette

smoke). Exposure to styrene has been estimated to be six times higher for smokers than

for nonsmokers (Cohen et al. 2002). As noted in Table 2-10, Fishbein (1992) estimated a

styrene exposure of 400 to 960 μg/day based on 20 cigarettes per day and inhalation of

20 to 48 μg of styrene per cigarette. Tang et al. (2000) estimated an additional styrene

intake (above the daily intake from air and food) of 100 μg/day, based on 20 cigarettes

per day and inhalation of 5 μg styrene per cigarette. In a study on toxic compounds in

ETS, Bi et al. (2005) presented styrene levels in ETS for three types of cigarettes: ultra

low tar (146 μg/cigarette), full flavor low tar (159 μg/cigarette), and full flavor (119

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μg/cigarette). Charles et al. (2007) found similar levels in ultra-low–nicotine (90

μg/cigarette), standard nicotine (160 μg/cigarette), and low nicotine (162 μg/cigarette)

cigarettes. Miller et al. (1998) assessed the contribution of ETS to total styrene exposure.

The results of this study showed that for the study population, 15% of a passive smoker’s

and 8% of a non-smoker’s daily intake of styrene was attributable to ETS. (A passive

smoker does not smoke but spends at least some time in a closed area with a smoker.)

Charles et al. (2007) found that side-stream smoke emissions greatly exceeded main-

stream smoke emissions. Analysis of emissions from a low-nicotine cigarette showed

main-stream smoke emissions of 11 μg/cigarette and side-stream smoke emissions of 147

μg/cigarette.

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Exposure to styrene from food ingestion has been estimated in a number of studies.

Lickly et al. (1995b) estimated U.S. dietary styrene exposure at 9 μg/day. A study of

residents of the United Kingdom in 1983 showed styrene intake from food ingestion of 1

to 4 μg/day (Lickly et al. 1995b). Another study of U.K. residents, which employed a

probabilistic modeling approach, estimated median daily intake of styrene from food

contaminated with food contact materials to be 0.039 μg/kg b.w. per day for adults, 0.048

μg/kg b.w. per day for youths, and 0.035 μg/kg b.w. per day for seniors (Holmes et al.

2005). Another study, based on the average per capita consumption figures of the general

population in Germany, estimated the average annual styrene intake via food

consumption to be roughly 0.8 to 4.5 mg/person [2.2 to 12.3 μg/d] (Tang et al. 2000).

Using the same data and applying a U.S. FDA consumption factor based on the

assumption that only 10% of foods are packaged in polystyrene, an annual intake of 0.08

to 0.45 mg/person [0.22 to 1.23 μg/d] was estimated. Other studies have estimated annual

per-person styrene intake via food ingestion ranging from 0.26 to 14.8 mg [0.7 to 40.5

μg/d] (Tang et al. 2000).

In an exposure and risk assessment for styrene, Cohen et al. (2002) based their

assessment only on inhalation and food ingestion exposures, assuming that exposure from

ingestion and dermal contact with water is negligible due to its limited occurrence and

low levels. Estimated airborne concentrations for this study are presented in Table 2-12.


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Table 2-12. Estimated annual and lifetime exposures for the general public

Exposure scenario

Maximum annual average

(ppb) Lifetime average

(ppb) Typical ambient exposure 1 1

High-end ambient exposure 5 5

Exposure to styrene from smoking 6 < 6

Living 100 m from a 100,000-lb/yr emission facility (high-exposure scenario, 95th percentile individual)

12 2.8

Living at the point of greatest exposure in the vicinity of a 1 million-lb/yr emission facility (high-exposure scenario, 95th percentile individual)

700 219

Source: Cohen et al. 2002.

In assessing exposure from food, the authors first estimated exposure from naturally

occurring substances. Using upper-end concentration data from the literature the authors

estimated that exposure for the U.S. population would be less than 0.2 μg/kg of food

ingested. They then assumed food consumption of 3 kg/day and arrived at a daily styrene

ingestion rate of 0.6 μg/day. The authors used the exposure level of 9 μg/day presented

by Lickly et al. (1995b) (see above) for ingestion of food contaminated through migration

from polystyrene packaging. The authors concluded that 10 μg/day is a reasonable upper

bound estimate for total dietary intake, which they noted corresponds to 0.2 μg/kg b.w.

for a 70-kg adult.

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Several studies have confirmed styrene exposure to the general public through the use of

biological monitoring. In one study, styrene was detected in all eight human breast milk

samples from women in four U.S. cities (Howard 1989). In a National Human Adipose

Tissue Survey by the U.S. EPA in 1982, styrene was detected in wet adipose tissue with a

frequency of 100% at concentrations ranging from 8 to 350 ppb. Styrene also has been

detected in the general population in blood at a mean concentration of 0.4 μg/L and in

exhaled breath at mean concentrations of 0.7 to 1.6 μg/m3 (ATSDR 1992).

Blood styrene levels were assessed in the Priority Toxicant Reference Range Study

conducted as part of the Centers for Disease Control and Prevention’s (CDC) Third

National Health and Nutrition Examination Survey (NHANES III). The Priority Toxicant


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Reference Range Study assessed blood levels of numerous VOCs among a nonstatistical

subsample of NHANES III participants aged 20 to 59 (NCHS 2000). Samples in which

styrene was below the detection limit were assigned a value of 0.013 μg/L, which is equal

to the lower detection limit (0.019 μg/L) divided by the square root of 2. Of 624 samples,

styrene levels were below the detection limit in 78 samples (12.5%), and ranged from

0.019 to 4.006 μg/L in 546 samples. The mean styrene level for all 624 samples was 0.07

μg/L, the median was 0.04 μg/L, and the 95th percentile value was 0.18 μg/L (Ashley et

al. 1994, Sexton et al. 2005). [The means obtained by assigning a value of 0.013 μg/L to

samples below the detection limit or by assigning a value of 0.00 μg/L to these samples

were the same after rounding.] It is important to note that because this study was

conducted with a nonstatistical subsample of NHANES III participants, statistical

weights cannot be assigned, and estimates for the total U.S. population therefore cannot

be calculated (NCHS 2000).

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Sexton et al. (2005) examined blood levels of styrene and several other VOCs over a

two-year period in more than 150 children from two poor, minority neighborhoods in

Minneapolis, MN. For styrene, the mean concentration was 0.17 µg/L, the median

concentration was 0.12 µg/L, and the 95th percentile concentration was 0.50 μg/L. The

authors compared these levels with the NHANES styrene levels for adults (0.07 for

mean, 0.04 for median, 0.18 for 95th percentile) and noted that the elevated levels of

styrene in children were unexpected. The authors noted that the children’s VOC

exposures and related blood levels were the product of concentrations in the air, water,

soil, dust, food, beverages, and consumer products with which they came into contact

through everyday activities and behaviors, but they were unable to explain the elevated

styrene levels for the children. The authors made note of the fact that the NHANES data

included smokers, which made the elevated styrene levels in children even more

surprising, and they ultimately concluded that the source of the children’s exposure to

styrene needed further investigation. In a follow-up study, Sexton et al. (2006) measured

blood levels of several chemicals, including styrene, in 43 children aged 3 to 6 from a

socioeconomically disadvantaged neighborhood in Minneapolis, MN. The mean and


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median for the group was 0.07 ng/mL (μg/L) and the 95th percentile was 0.11; levels that

the authors noted were similar to the NHANES levels.

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2.5 Occupational exposure 3 In some workplace settings, styrene air levels can exceed by several orders of magnitude

the levels generally found in outdoor and indoor air. [Because of this, air levels in this

section are presented in parts per million [ppm] rather than parts per billion [ppb], which

were used in the outdoor air and indoor air sections above.] Workers in a number of

different industries can be exposed to styrene. Workers can be exposed during the

production and use of styrene monomer, polystyrene, glass fiber–reinforced plastics,

styrene-butadiene rubber and other styrene-based polymers, and in other miscellaneous

occupations (ATSDR 1992, IARC 2002). The National Occupational Hazard Survey,

conducted by the National Institute for Occupational Safety and Health (NIOSH) from

1972 to 1974, estimated that 292,018 employees were occupationally exposed to styrene

at 16,394 facilities. The National Occupational Exposure Survey, conducted by NIOSH

from 1981 to 1983, estimated that 333,212 employees (including 86,902 women) were

occupationally exposed to styrene at 24,702 facilities in 154 industries. The U.S. Bureau

of Labor Statistics (BLS) uses the Standard Occupational Classification (SOC) system to

classify workers into occupational categories for labor statistics analyses. Workers are

classified into one of over 820 occupations according to their occupational definitions. In

May 2006, the BLS estimated that 32,510 workers were employed in SOC code 51-

2091 — Fiberglass Laminators and Fabricators (defined as “laminate layers of fiberglass

on molds to form boat decks and hulls, bodies for golf carts, automobiles, or other

products”). “Ship and boat building” was the largest subcategory in this SOC segment,

with 12,910 employees (BLS 2007). No information was found on the numbers of

workers in the other industrial segments mentioned above.

Based on the breakdown of industrial sectors used for the review of the human cancer

data in Section 3, this section provides information on the following three major

industrial settings: the reinforced-plastics industry, the styrene-butadiene rubber industry,

and the styrene monomer and polymer industry. The section concludes with a discussion

of other miscellaneous occupational exposures. Section 3 of this document reviews


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epidemiologic studies from the United States and other countries; therefore, this section

reports information identified for occupational exposures either in the United States or in

other countries.

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2.5.1 The reinforced plastics industry 4 IARC (2002) has noted that the highest occupational exposures to styrene, with respect to

the number of employees and exposure levels, occur in the fabrication of objects such as

boats, car and truck parts, tanks, tubs, and shower stalls from glass fiber–reinforced

polyester composite plastics. [“Reinforced plastics” is the term generally used in this

document, but other terms used to describe this industry include fiberglass-reinforced

plastics, fiberglass-reinforced polyester resin, reinforced plastic composites, and

laminated plastics.] Styrene has been noted to be the principal VOC present in resins used

in the reinforced plastics industry (Hillis 1997, Hillis and Davis 1995, MnTAP 2007,

Säämänen 1998), and according to the U.S. EPA, styrene is the main hazardous air

pollutant in the reinforced plastic composites industry (EPA 2003). Table 2-13 at the end

of this section provides both styrene air levels and levels of biological markers for the

studies where they were assessed. The text discusses the major issues related to these

studies. This section presents information on worker exposures in the reinforced plastics

industry. Because much of the discussion on exposure levels involves process and job

descriptions, the section begins with an overview of two of the main processes used in the

production of glass fiber–reinforced plastic products. This is followed by historical

industry-wide exposure levels, levels based on the product being manufactured or the

manufacturing process employed, and levels based on specific jobs or tasks. Studies that

assessed respirator use are then discussed briefly followed by a short discussion of

studies that measured styrene-7,8-oxide concurrently with styrene. The section concludes

with a discussion of studies that have assessed dermal exposure in the reinforced plastics

industry.

2.5.1.1 Process description 27 Two main processes are used to produce glass fiber–reinforced plastic composite

products: an open-mold process and a closed-mold process. In general, large glass fiber–

reinforced plastic composite products are built using an open-mold process. With this


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process, a mold of the desired final product is sprayed with a layer of gel coat, which is

pigmented polyester resin that hardens and becomes the smooth outer surface of the

product (CDC 2007, EPA 1997a). After the gel coat has hardened, it is coated with a

“skin coat” of chopped glass fibers and polyester resin and then rolled with a roller to

compact the fibers and remove air bubbles. After the skin coat has hardened, additional

layers of fiberglass cloth and chopped glass fibers saturated with resin are added until the

desired final thickness is obtained. These layers of resin and chopped glass fibers are

usually applied with either spray equipment (spray-up), such as a chopper gun, or by

hand using a bucket and brush or paint-type roller (lay-up). The layers are compressed by

rolling the surface, usually by hand. After the resin has cured, the part is removed from

the mold and the edges are trimmed to the final dimensions. Exposure to styrene can

occur at all steps of the open-mold process, as both the gel coat and the polyester resin

contain substantial levels of styrene.

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Closed molding is the name given to fabrication techniques in which reinforced plastic

parts are produced between the halves of a two-part mold or between a mold and a

flexible membrane (EPA 1997a). There are a number of different processes that are

considered a closed-mold process. One example, called “resin transfer molding,” uses

half molds that are closed before resin injection and curing, thereby potentially reducing

exposure during this stage (CDC 2007, EPA 1997a). However, prior to injection of the

resin, the process is similar to the open-mold process. First, a gel coat is applied to the

interior surface of both molds to provide a smooth finish on all external surfaces after the

cure. Following application of the gel coat, dry fiber reinforcement mat is placed into the

mold before closing. After the mold is closed, resin and initiator are pumped into the

mold cavity by a pressure pump. Curing takes place while the mold is closed. While it is

expected that exposures will be limited during the final resin transfer and curing inside

the closed mold, the closed-mold process does not control emissions and potential

exposures during gel-coat application. In this process, gel coating is generally done inside

a spray booth. Usually, one worker sprays the gel coat inside the spray booth, and another

worker applies the fiberglass inside the mold, closes the mold, and sets up the mold for

resin injection. Both of these jobs have the potential for exposure to styrene. Other

closed-mold processes include vacuum bagging, vacuum-assisted resin transfer molding,


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and compression molding (EPA 1997a). The common feature of these processes is that at

least part of the process occurs in a closed system, thereby potentially allowing for the

control of styrene emissions and exposure.

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2.5.1.2 Historical industry-wide exposure levels 4 Historically, the highest styrene exposure levels for reinforced-plastics workers has been

in the range of several hundred parts per million, although declining levels have been

reported to have occurred over the past several decades. In a study published in 1981 of

12 plants manufacturing fiberglass in Washington state, 40% of 8-hour samples contained

styrene at over 100 ppm (IARC 2002). Kolstad et al. (1994) presented styrene exposure

level data based on 2,473 personal air samples taken at workplaces in Denmark between

1964 and 1988 (see Section 3.1.4). Mean styrene levels were 180 ppm for 1964 to 1970,

88 ppm for 1971 to 1975, and 43 ppm for 1976 to 1988. In an extension of the 1994

study, Kolstad et al. (2005) used data from 2,454 personal measurements of airborne

styrene taken by the Danish National Institute of Occupational Health between 1960 and

1996 to develop a semi-quantitative method to assess occupational exposure to styrene in

the reinforced plastics industry when individual data are not available. Calendar year was

reported to be a strong and consistent predictor of styrene exposure levels; along with

product produced (boats) and process (hand and spray lamination). For the time period

between 1960 and 1990, styrene exposure levels were reported to have declined by 7%

annually. Kolstad et al. calculated exposure scores for individuals based on estimated

exposure probability and exposure levels. Styrene exposure scores for 1,519 subjects

based on short-term and long-term samples are presented in Figures 2-4 and 2-5 below.

Exposure scores declined by about 10-fold from the 1960s to the 1990s, and the authors

noted that this reflected a decline in styrene exposure levels. This study did not assess

dermal exposures.

Similarly, Kogevinas et al. (1994a) reported that in Denmark, average exposure levels

among laminators were about 200 ppm in the late 1950s, about 100 ppm in the late

1960s, and about 20 ppm in the late 1980s (see Section 3.1.5). In a review of 16 studies

by Pfäffli and Säämänen (1993), a similar temporal decline in exposure levels was seen


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in air concentration data from the United States, Canada, Japan, and Europe from the

1950s through 1992.

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Figure 2-4. Temporal decline in styrene exposure scores (short-term samples [< 1 h]) estimated for reinforced plastics workers

Source: Kolstad et al. 2005.

Jensen et al. (1990) presented data based on 2,528 measurements of styrene at 256

workplaces in Denmark between 1955 and 1988. Annual mean concentrations decreased

from a high of 1,005 mg/m3 [236 ppm] in 1964, to a low of 88 mg/m3 [21 ppm] in 1988.

Period-specific mean concentrations were 714 mg/m3 [168 ppm] for 1955 to 1970, 274

mg/m3 [64 ppm] for 1971 to 1980, and 172 mg/m3 [40 ppm] for 1981 to 1988. For the

entire 1955 to 1988 period, the mean concentration was 265 mg/m3 [62 ppm].

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Figure 2-5. Temporal decline in styrene exposure scores short-term samples [< 1 h]) estimated for reinforced plastics workers

Source: Kolstad et al. 2005.

Serdar et al. (2006) noted that in general, air levels of styrene (and styrene-7,8-oxide)

appear to have decreased substantially in this industry from the 1980s through the early

2000s. Although styrene exposures have been reduced substantially through improved

work practices and products (Kolstad et al. 1994), Miller et al. noted in 1994 that peak

concentrations could still exceed 100 ppm, especially during the manufacture of large

items, and this can be seen in Table 2-13 for measurements taken through the 1990s.

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2.5.1.3 Exposure levels based on product or manufacturing process 7 Several factors influence the level of styrene in workplace air in the reinforced plastics

industry. Chief among these are the surface area of the product being manufactured, and

the manufacturing process used. In general, the manufacture of products with large

surface areas, such as boats, truck parts, and shower stalls, by the open-mold process

results in higher exposures than manufacture of smaller products by a closed-mold

process. Lemasters et al. (1985) found average styrene exposure levels associated with


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open-mold processes (24 to 82 ppm) to be 2 to 3 times those associated with closed-mold

processes (11 to 26 ppm).

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[There does not appear to be a clear distinction as to what products result in the highest

exposure levels.] Although boat building has often been associated with higher levels,

this has not always been the case. IARC (2002) reported that boat building involves

higher styrene exposures than any other industrial sector. Kolstad et al. (2005) reported

that product (boats), process (lamination), and calendar year were the major determinants

of styrene exposure, and the authors noted that styrene exposure levels were higher by a

factor of 1.6 to 1.7 at companies producing boats than at companies producing other

products. As discussed below, other studies often reported higher levels for the

manufacture of products other than boats.

A survey by the State of California Division of Occupational Safety and Health ranked

the highest worker exposure levels by industry based on geometric mean exposure levels.

Tub or shower-stall manufacture was highest at 53.6 ppm, followed by camper

manufacturing (41.0 ppm), boat manufacturing (29.1 ppm), spa manufacturing (25.8

ppm), miscellaneous manufacturing (22.0 ppm), and tank manufacturing (12.7 ppm).

In an assessment of 328 fiberglass-reinforced plastics workers in 13 similar sized plants

in the Pacific Northwestern United States, Serdar et al. (2006) noted that exposures to

styrene varied greatly based on the product being manufactured. Air levels of styrene

decreased with product categories in the order of RVs > pipe and tank > hot tub > boat

building ~ truck manufacture. In an assessment of 17 U.S. reinforced plastics workplaces,

Luderer et al. (2004) reported mean air levels by products manufactured in the order of

truck and RV > bathtub > pipe, tank > boat.

2.5.1.4 Exposure levels based on job or task 24 Exposure to styrene also varies with the type of job or task performed. (See Section 2.6

for a description of biological indices used to measure styrene exposure.) Using 4,689

urine samples obtained from reinforced plastics workers in the region of Emilia

Romagna, Italy, Galassi et al. (1993) found that hand laminators had the highest mean

mandelic acid (MA) levels (682 mg/g creatinine), followed by spray laminators (404


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mg/g creatinine), rollers (327 mg/g creatinine), semiautomatic process operators (243

mg/g creatinine), and non-process workers (186 mg/g creatinine). Similarly, mean

styrene air levels were highest for hand laminators (227 mg/m3 [53 ppm]); however,

rollers were exposed to the next highest levels (163 mg/m3 [38 ppm]), followed by spray

laminators (134 mg/m3 [31 ppm]), and semiautomatic process operators (85 mg/m3 [20

ppm]). The authors noted a clear positive correlation between air levels and urinary

mandelic acid (MA) levels. The authors also noted that air levels generally decreased

with time.

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Based on U.S. data published in 1985, IARC (2002) reported mean personal breathing-

zone air concentrations for four boat fabrication tasks as follows: hull lamination, 331

mg/m3 [77 ppm]; deck lamination, 313 mg/m3 [73 ppm]; small parts lamination, 193

mg/m3 [45 ppm]; and gel coating, 202 mg/m3 [47 ppm]. Measured concentrations across

all jobs or tasks ranged from 7 to 780 mg/m3 [1.6 to 183 ppm].

Based on results from one study that assessed 8-hour TWA exposure levels across job

categories and tasks within the reinforced plastics industry, spray-up/lay-up operators had

the highest exposure levels with a mean of 256 mg/m3 [60 ppm] and a range of 21 to 511

mg/m3 [5 to 120 ppm] (IARC 2002). For other jobs, which included gel coating and 7

other job categories, the mean exposure levels ranged from ≤ 43 to 192 mg/m3 [≤ 10 to

45 ppm], and overall exposure levels ranged from 0 to 362 mg/m3 (0 to 85 ppm). Based

on a study of 237 workers in 30 Finnish reinforced plastics plants, Nylander-French et al.

(1999) reported that the highest 8-hour TWA styrene exposure level across 6 categories

of tasks was for hand lamination of large objects (156 mg/m3 [37 ppm]) and that these

levels were approximately 4-fold higher than exposure levels for foremen (42.6 mg/m3

[10 ppm]), which was the group with the lowest exposure levels. Overall, the mean 8-

hour TWA concentration of styrene was 122 mg/m3 [28.6 ppm] with a range of 3.2 to 608

mg/m3 [0.75 to 142.7 ppm].

In an assessment of 48 workers in a U.S. reinforced plastics boat manufacturing facility,

Rappaport et al. (1996) grouped workers into 10 categories based on the job performed.

They reported that spray operators had the highest exposure levels with a mean of 141


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mg/m3 [33 ppm] while laminators had the second highest levels at 130 mg/m3 [30.5

ppm]. Overall, the mean styrene air level was 64.3 mg/m3 [15.1 ppm] with a range of

0.978 to 235 mg/m3 [0.23 to 55.14 ppm].

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In Finnish factories that produced boats, car parts, and building materials from polyester-

based reinforced plastics, average styrene concentrations in personal air samples were

133 ppm for hand applicators and 130 ppm for spray applicators (IARC 1994b). Based on

2,528 measurements of styrene at 256 workplaces in Denmark between 1955 and 1988,

Jensen et al. reported that the work processes associated with the highest concentrations

were spray-up and unspecified lay-up operations.

Fairfax and Swearngin (2005) reported the results of a 2003 planned inspection by OSHA

of a facility that produced bathtubs and shower stalls by spray application of styrene-

based gel coat and polyester resin. The OSHA inspection consisted of full-shift personal

air monitoring of two gel-coat operators and two chopper gun operators and showed

TWA styrene levels ranging from 64 ppm (chopper gun operator) to 318 ppm (gel-coat

operator). Follow-up measurements (personal and area) were made in June and October

of 2003. June TWA levels ranged from 66 ppm to 100 ppm (both were gel-coat

operators), and October levels ranged from 45 ppm (gel-coat operator) to 110 ppm

(chopper gun operator). Ultimately, switching to a product containing less styrene and

operational changes were needed to reduce exposure levels below the regulatory limit of

100 ppm.

2.5.1.5 Studies assessing respirator use 21 Nakayama et al. (2004) evaluated the efficiency of various types of respiratory protective

equipment by comparing styrene exposure levels to urinary levels of mandelic acid

among 39 workers in 5 fiberglass-reinforced plastics factories. For the 39 workers, the

area monitoring results ranged from not detected (< 0.5 ppm) to 67.4 ppm. (The authors

noted, however, that in a gel-coating operation that used an agent containing 40% to 50%

styrene, styrene levels as high as 2,000 ppm were present for short periods of time.)

Personal monitoring levels for the 39 employees ranged from 0.7 ppm to 318.8 ppm, and

creatinine-adjusted mandelic acid levels ranged from 10 to 1,606 mg/g creatinine. The

authors concluded that the efficiency of disposable gauze type and dust-proof respirators


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was nearly zero and that the efficiency of half-mask respirators was highly dependent on

the frequency of cartridge replacement.

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Inaoka et al. (2002) conducted a study to examine winter-summer levels and associations

in airborne styrene exposure concentrations and end-of-shift urinary mandelic acid levels,

and the protective effect of disposable particulate respirators containing charcoal fiber

(charcoal mask) and a charcoal granule cartridge mask (gas mask). The study was

conducted in the winter of 1997 to 1998 and involved 105 workers in 10 small-sized

fiberglass-reinforced plastics production facilities in Japan. Airborne styrene was

measured using passive samplers attached to the workers’ collars, near the neck, and

urine samples were collected at the end of the shift for MA analysis. The authors

concluded that the charcoal mask provided little protection from styrene exposure, but

that the gas mask prevented 45% to 49% of styrene from being inhaled. The authors also

concluded that individual exposures to styrene and urinary mandelic acid levels did not

differ by season.

In a study to assess the capacity of negative-pressure half-mask respirators to protect

workers from styrene exposure, personal sampling of styrene air levels and urinary

styrene levels was performed on seven fiberglass-reinforced plastics workers over two

successive weeks: one week without respirators and the following week with respirators

(Gobba et al. 2000). During the study period, mean TWA workplace air concentrations of

styrene were estimated for Monday, Wednesday, and Friday for both morning shifts and

afternoon shifts. These six mean TWA air concentrations ranged from 169.2 to 335.7

mg/m3 [39.6 to 78.6 ppm] with a range across all measurements of 70.9 to 488.1 mg/m3

[16.6 to 114.5 ppm]. Mean urinary styrene levels ranged from 80 to 96.1 μg/L without

the use of respirators and from 31.5 to 47.2 μg/L with the respirators. The estimated

reduction of urinary styrene levels due to the respirators ranged from 30% to 90% with a

mean of 60%.

2.5.1.6 Styrene-7,8-oxide exposures 27 Workers in the reinforced plastics industry can potentially be exposed to styrene-7,8-

oxide as well as styrene, and several studies have measured exposure levels for both


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substances showing styrene levels at two to three orders of magnitude higher than

styrene-7,8-oxide levels. Serdar et al. (2006) (discussed above) noted that styrene levels

in full-shift personal breathing-zone samples were roughly 500-fold higher than styrene-

7,8-oxide levels.

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For 237 workers in 30 Finnish reinforced plastics plants, the mean 8-hour TWA

concentration of styrene was 122 mg/m3 [28.6 ppm] with a range of 3.2 to 608 mg/m3

[0.75 to 142.7 ppm], while the mean concentration of styrene-7,8-oxide was 0.183 mg/m3

[0.04 ppm] with a range of 0 to 0.883 mg/m3 [0 to 0.21 ppm] (Nylander-French et al.

1999). The authors found that styrene-7,8-oxide levels were positively correlated with

styrene exposure levels.

In a boat manufacturing factory in the United States, Rappaport et al. (1996) reported a

mean styrene air level of 64.3 mg/m3 [15.1 ppm] with a range of 0.978 to 235 mg/m3

[0.23 to 55.14 ppm] and a mean styrene-7,8-oxide level of 0.159 mg/m3 [0.037 ppm)]

with a range 0.0134 to 0.525 mg/m3 [0.003 to 0.12 ppm]. IARC (1994b) reported that for

the 19 most heavily exposed workers in a boat manufacturing company, the mean styrene

exposure level was 64 mg/m3 [15 ppm] while the mean styrene-7,8-oxide level was 0.14

mg/m3 [0.03 ppm]. In Finnish factories that produced boats, car parts, and building

materials from polyester-based reinforced plastics, average styrene concentrations in

personal air samples were 133 ppm for hand applicators and 130 ppm for spray

applicators; the corresponding average styrene-7,8-oxide levels were 0.04 ppm and 0.12

ppm.

Table 2-13 presents styrene levels in ambient air in the reinforced plastics industry. For

this table and the remainder of the tables presenting occupational exposure levels, the

number of samples is presented when it was available in the referenced source.


Table 2-13. Summary of measured styrene exposure levels in the reinforced plastics industry.

Industrial segment (year measured)

Specific job, process, or production area

Styrene air levels Mean (range)

(ppm) Biological levels

Mean (range) Reference (Location) Boat, hot tub, pipe and tank, RV, and truck mfg. (1996–1999)

Tasks across all categories Production area boat building hot tub pipe and tank RV truck

9.14a (< 1–117) (N = 328) 4.41a (< 1.0–68.6) (N = 138) 6.85a (< 1.0–62.9) (N = 13) 16.0a (1.67–79.0) (N = 50) 45.1a (6.74–117) (N = 48) 4.22a (< 1.0–46.3) (N = 76)

SB: 0.083 (< 0.001–2.05) mg/L (N = 295) SOB: 0.069 (< 0.05–0.135) μg/L (N = 212)

Serdar et al. 2006

(USA)

Small sized facilities, products not specified (1997–1998)

Numerous tasks including hand/spray laminators, rollers, semiautomatic process workers, and non-process workers

10.3–35.9 (NR) MA: 70-350 mg/g Inaoka et al. 2002

(Japan)

Industry wide (1960–1996)

Overall short-termb Product boats other products

Task hand or spray lamination other

Year before 1970 1970–1974 1975–1979 1980–1989 1990–1996

59.7 (ND–639)c (N = 2,208) 100.5 (ND–639)c (N = 670) 42.0 (ND–563)c (N = 1,537) 61.0 (ND–639)c (N = 2,074) 39.0 (0.7–177)c (N = 133) 173.3 (11.7–639)c (N = 113) 94.2 (2.3–587)c (N = 425) 71.9 (0.9–403)c (N = 360) 41.1 (ND–456)c (N = 954) 19.8 (0.2–171)c (N = 355)

– Kolstad et al. 2005

(Denmark) [note that this study is an extension of Kolstad et al. 1994 presented below]

Boat mfg. (NR) hand-spraying lamination 8.71 (0.47 to 126) (N = 45)

MA+PGA: 300 (10.2 to 1,856) mg/g CR (N = 95)

PHEMA: 0.9 (0.01 to 3.29) mg/g CR (N = 45)

Migliore et al. 2006a

(Italy)

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Mean (range) Reference (Location) VPT: 1.9 (0.1 to 7.74) mg/g CR (N = 45)

Tub/shower mfg. (2001–2003)

chopper gun and gel coating operations

34–318 (NR) (N = 49) – Fairfax and Swearngin 2005

(USA) Boat mfg. (1998) not specified 52.3 (0.3–133.5) (N = 73) MA: 288.5 (1.0–1,813.2) mg/g CR (N =

73) PGA: 123.8 (4.4–481.5) mg/g CR (N = 73)

Ma et al. 2005

(Japan)

Not specified (1998–1999)

low-exposure jobs (N = 55) high-exposure jobs (N = 53)

4.1 (0.07–22.5)c 9.3 (1.1–13.3)c (LWAE) 3.7 (0.07–15.1)c 22.6 (13.6–30.2)c LWAE

MA: 1.0 (0.1–2.7) mmol/g CR [152 (15.2–410) mg/g CR]

MA: 0.8 (0.1–2.1) mmol/g CR [121.6 (15.2–319) mg/g CR]

Iregren et al. 2005a

(Sweden)

Boat mfg. (NR) fibrous glass department lamination department

42.5 (7.27–84.7) (N = 53)

71.6 (10.32–183) (N = 67) – Okun et al. 1985, Ruder

et al. 2004

(USA) Various types of products (NR)

various processes NR (ND–67.4) (area) (N = 29)

NR (0.7–318.8) (personal) (N = 39)

MA: NR (10–1,606) mg/g-creatinine (N = 39)

Nakayama et al. 2004

(Japan)

Boat, tub, truck/RV, pipe/tank mfg., and boat repair (NR)

not specified 9a (< 1–142) (N = 402) SB: 0.0089 (< 0.001–2.05) mg/L (N = 302)

Luderer et al. 2004

(USA)

Tubs/showers, sheet paneling, and other unspecified products at 4 facilities (NR)

open-mold, closed-mold, and press-methods using spray/chopper guns, sheet press, and hand lay-up and die molding

9.2–55 (0.1–140.3) (N = 99) MA: 190–1,740 (< 10–6,980) mg/g CR PGA: 80–490 (< 10–2,250) mg/g CR (N = 104 for both MA and PGA)

Dalton et al. 2003, Lees et al. 2003

(USA)

Boats, tanks, various processes and jobs 1.1–15.6d (NR) MA: 24.6–227 (NR) mg/g CR Liljelind et al. 2003


9/29/08 57





Mean (range) Reference (Location) bathroom fixtures (NR)

(N = 12 workers with 3 to 4 matched samples)

(Sweden)

Industry-wide large open-mold spray-up/lay-up operations (NR)

Overall Product specific mfg.

tub/shower camper boat spa miscellaneous tank

43 (0.2–288) 53.6d (NR) 41.0d (NR) 29.1d (NR) 25.8d (NR) 22.0d (NR) 12.7d (NR)

– IARC 2002

(USA)

Boat mfg. (NR) hull lamination deck lamination small parts lamination gel coating

77.7 (1.64–183) (N = 168)

73.4 (12.2–160) (N = 114) 45.3 (7.98–130) (N = 70)

47.4 (5.4–103) (N = 45)

– IARC 2002

(USA)

Not specified (1967–1978)

spray-up/lay-up 8 other job categories

60e (5–120) ≤ 10–45 (0–85)

– IARC 2002

(USA) Not specified (NR)

hand rolling, spraying, finishing

39.6–78.6 (16.6 to 114.5) [N = 84]

SU: 31.5–96.1 (7.4–133.3) μg/L [N = 41]

Gobba et al. 2000

(Italy) Various industries, primarily reinforced-plastics production (1973–1983)

primarily laminators MA: 2.3a (0–47) mmol/L [350 (0–7,144 mg/L)] (N = 10,336)

Anttila et al. 1998

(Finland)

Boats, containers, pipes and tubes, small parts, sheets, and vehicle parts (1988–1990)

Jobs hand lamination, large objects

hand lamination, small objects

spraying and gel coating automated lamination

36.6 (NR)c [N = 216] 35.2 (NR)c [N = 98] 30.5 (NR)c [N = 22] 13.1 (NR)c [N = 46]

– Nylander-French et al. 1999

(Finland)


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Mean (range) Reference (Location) assembly and mold preparation

foreman Products boats small and form parts sheets, elements, car parts containers and tubes

11.7 (NR)c [N = 56] 10.0 (NR)c [N = 28] 30.97 (NR)c [N = 274] 31.44 (NR)c [N = 68] 21.19 (NR)c [N = 78] 24.87 (NR)c [N = 56]

Boat mfg. (1987–1988)

laminator (16 workers) service (6 workers) mold repair (3 workers) patcher (8 workers) painter (6 workers) spray operator (1 workers) mechanic (4 workers) deck rigger (2 workers) assembly (1 workers) supervisor (1 workers)

30.5 (NR)c 6.55 (NR)c 27.45 (NR)c 3.19 (NR)c 6.5 (NR)c 33.08 (NR)c 1.95 (NR)c 0.99 (NR)c 0.41 (NR)c 5.89 (NR)c (N = 2 to 7 samples per worker)

– exhaled styrene = 1.76 (0.007– 8.12) mg/m3 (N = 1 to 7 measurements per worker)

Industry-wide (1964–1970) (1971–1975) (1976–1988)

not specified (N = 2,473 personal air samples 1964–1988)

180 (NR) 88 (NR) 43 (NR)

– Kolstad et al. 1994

(Denmark)

Industry-wide overall TWA concentrations spray-up/lay-up operators TWA

1–200 60e (5–120)

– Wong et al. 1994

(USA)

Industry-wide (NR)

Process workers hand laminators rollers spray laminators semiautomatic process

48c (NR) (N = 1,305) 53c (NR) (N = 1,028)

38c (NR) (N = 40) 31c (NR) (N = 166) 20c (NR) (N = 71)

MA: 631 (NR) mg/g CR (N = 2,820) MA: 682 (NR) mg/g CR (N = 2,386) MA: 327 (NR) mg/g CR (N = 63) MA: 404 (NR) mg/g CR (N = 250) MA: 243 (NR) mg/g CR (N = 121)

Galassi et al. 1993

(Italy)


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Mean (range) Reference (Location) operators

Non-process workers

16.7c (NR) (N = 159) MA: 186 (NR) mg/g CR (N = 762)

Industry-wide (1955–1970) (1971–1980) (1981–1988) (1955–1988)

not specified 168 (NR) (N = 227) 64 (NR) (N = 1,117) 40 (NR) (N = 1,184) 62 (21–236) (N = 2,528)

– Jensen et al. 1990

(Denmark)

Industry-wide (1969–1981)

open-mold press-mold

3–82 (NR) (N = 1,084) 4–26 (NR) (N = 402)

– Lemasters et al. 1985

(USA) CR = creatinine; LWAE = lifetime weighted average exposure; MA = mandelic acid; mfg. = manufacturing; NA = not assessed; ND = not detected; NR = not reported; PGA = phenylglyoxylic acid; PHEMA = phenylhydroxyethylmercapturic acids; SB = blood styrene level; SU = urinary styrene level; VPT = vinylphenols; SOB = blood styrene-7,8-oxide level. aMedian. bData also presented in source document for long-term samples showing levels that were generally around one-half the levels reported here. cPresented in mg/m3 in source document. dGeometric mean(s). eReported as typical level.


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2.5.1.7 Dermal exposure 1 [The potential exists for dermal exposure in the workplace to styrene or styrene-

containing materials in either aqueous or vapor form. While dermal exposure can occur in

any industry that uses styrene, the potential is especially high in the reinforced-plastics

industry during lamination operations and is thus discussed in this section.]

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In a study assessing the potential routes of exposures to styrene in the glass fiber–

reinforced-plastics industry, workers were equipped with various types of protective

equipment: total protection with an insulating suit and mask, respiratory equipment only,

skin protection only, and no protection (Limasset et al. 1999). Urinary styrene excretion

levels did not differ significantly between the group with total protection and the group

with only respiratory equipment. The authors concluded that percutaneous absorption

was not a particularly important pathway for styrene absorption in the glass fiber–

reinforced polyester industry. These results were similar to those of Brooks et al. (1980).

Although Limasset et al. and Brooks et al. concluded that dermal absorption was not a

particularly important pathway for styrene exposure, Brown (1985) noted that when

factors such as skin hydration and its condition, individual and anatomical site variations,

and the permeability-enhancing effects of other compounds are considered, skin

absorption can be seen as a significant exposure route for pollutants. Luderer et al. (2005)

similarly noted that although some studies reported limited skin absorption of styrene in

workers, prolonged and repeated exposure to liquid styrene could result in exposures

equivalent to the lower range of doses received by inhalation.

In an experiment to assess skin absorption of the hand and forearm of liquid styrene or

styrene in aqueous solution, Dutkiewicz and Tyras (1968) reported that very short

exposure of the hands to liquid styrene (a few minutes) or longer exposure (about one

hour) to styrene in aqueous solution can result in the absorption of as much styrene as an

8-hour average air concentration of 0.05 mg/L [50 mg/m3 or 11.7 ppm]. They also noted

that urinary mandelic acid does not provide a reliable index of absorption if there is

simultaneous skin and lung exposures.

Eriksson and Wiklund (2004) used a patch sampling technique to study potential dermal

exposure to styrene in the glass fiber–reinforced-plastics industry. The legs, arms, and


9/29/08 61

upper back had the highest exposures. Potential total-body styrene exposure ranged from

544 to 17,100 mg/hour, with a geometric mean of 3,780 mg/hour. Wieczorek (1985)

investigated dermal absorption of styrene vapors in four volunteers exposed to styrene at

1,300 to 3,200 mg/m3 in a study chamber. The authors calculated that the dermal

absorption of styrene vapors contributed about 5% to the amount absorbed in the

respiratory tract under the same experimental conditions based on comparative mandelic

acid and phenylglyoxylic acid urinary levels. The authors presented a dermal vapor

absorption coefficient of 0.022 m3/hour based on the results of this study.

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Minamoto et al. (2002) performed patch tests on 29 workers (22 of whom had reported

having skin problems) employed in small-to-medium-sized reinforced-plastics plants in

Japan. The authors reported one positive test result for styrene.

See Section 5.1.1.1 for more information on dermal absorption of styrene.

2.5.2 The styrene-butadiene rubber (SBR) industry 13 Styrene-butadiene rubber is a copolymer of butadiene and styrene in which the styrene

units (approximately 25%) are distributed at random among butadiene units (75%) in

molecular chains (IISRP 1973). Styrene-butadiene rubber is the most widely used

synthetic rubber in the world, accounting for 46% of world consumption of synthetic

rubber and more than 26% of all rubber, natural or synthetic, in 2006 (ICIS 2008). Over

70% of styrene-butadiene rubber is consumed in the manufacture of tires and tire

products; however, non-tire uses are growing with applications including conveyor belts,

gaskets, hoses, floor tiles, footwear, and adhesives. This section provides a brief

overview of the two main styrene-butadiene rubber production processes (emulsion

process [Section 2.5.2.1] and solution process [Section 2.5.2.2]), followed by a discussion

of exposure levels that have been found within the styrene-butadiene rubber industry

(Section 2.5.2.3).

2.5.2.1 Emulsion process styrene-butadiene rubber production 26 The steps involved in synthetic rubber production include: (1) preparing the input

materials to the required form, (2) mixing the input materials together to react, (3)

stopping the reaction when the polymer chains have reached the appropriate length, (4)


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recovering any unused material, and (5) extracting and cleaning the rubber product

(IISRP 1973, Lattime 2000). Figures 2-6 and 2-7 provide process flow diagrams for the

polymerization and finishing stages within styrene-butadiene rubber production. Styrene,

butadiene, and other chemicals used in the production process are stored in tanks at the

production facility (tank farm) until they are pumped to reaction vessels. Butadiene is a

gas at normal temperature; however, it can be liquefied under pressure and is usually

handled in this form in the production of styrene-butadiene rubber. The butadiene,

styrene, water, emulsifier, and other materials are pumped into reaction vessels and

vigorously stirred to produce an emulsion. Current emulsion process methods employ a

cold production process whereby a combination of reducing and oxidizing agents are

used as catalysts: these catalysts are added to the first reaction vessel with the

styrene/butadiene mixture and polymerization begins immediately. Polymerization

continues as the emulsion passes through a series of reaction vessels. It is then brought to

a stop by the addition of a polymerization-inhibiting chemical called a shortstop.

Typically dimethyldithiocarbamate (DMDTC) is used as the shortstop. At this stage, the

rubber is in the form of minute rubber polymers suspended in the emulsion. The

shortstopped material is transferred to large vessels referred to as blowdown tanks, then

pumped into flash tanks where any unreacted butadiene is evaporated off, and then

pumped to a stripping column where unreacted styrene is removed by steam distillation.

At this point, the material is in the form of a relatively pure synthetic latex which is

accumulated in large storage tanks. Roughly 10% of all styrene-butadiene rubber is sold

in latex form for use as carpet backing, latex foam, and other products. While still in latex

form, extender oil and antioxidants may be added if extended rubber is being produced.

The latex is then passed into a tank where an acid brine is injected and the mixture is

stirred. During this process, the rubber coagulates in the form of a fine crumb, which is

then washed in fresh water, dewatered, and pressed into bales as a finished product.

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Figure 2-6. Typical continuous emulsion styrene-butadiene rubber polymerization process

Source: Lattime 2000

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Figure 2-7. Typical emulsion styrene-butadiene rubber finishing process Source: Lattime 2000

2.5.2.2 Solution process styrene-butadiene rubber production 1 The major difference between emulsion process, and solution process styrene-butadiene

rubber production lies in the co-polymerization chemistry. In contrast to the emulsion

process, where the feedstocks are suspended in a large proportion of water in the

presence of an initiator, the solution styrene-butadiene rubber copolymerisation process

proceeds in a hydrocarbon solution in the presence of an organometallic complex

(Lattime 2000). Solution styrene-butadiene rubber involves termination-free, anionic

polymerization initiated by alkyl lithium compounds, usually n-butyl lithium (NBL). The

use of alkyl lithium compounds is due to the solubility of this class of organometallics in

the hydrocarbon solvents (such as n-hexane or cyclohexane) that are used in the process.

Solution styrene-butadiene rubber production allows great variation in producing

different types of polymers. By adding certain chemicals, such as ethers, tertiary amines,

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and phosphates, random distribution of the co-monomers is achieved, making it possible

to control polymer composition, monomer distribution sequence, microstructure,

molecular weight, molecular weight distribution, and polymer chain structure. Lattime

(2000) noted that once the ability to control the randomization of solution styrene-

butadiene rubber was better understood and established, it began to displace some of the

market share of emulsion process styrene-butadiene rubber by allowing for more

variation and fine tuning of the styrene-butadiene rubber properties. Figure 2-8 provides a

process flow diagram for solution styrene-butadiene rubber production.

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8

Figure 2-8. Solution styrene-butadiene rubber manufacture by continuous process Bd=butadiene, HC=hydrocarbon, AO=antioxidant, and SS=shortstop.

Source: Lattime 2000

2.5.3 Styrene-butadiene rubber production exposure levels 9 Generally lower levels are seen in the styrene-butadiene rubber industry than the glass

fiber–reinforced-plastics industry, although significant exposures to workers can still

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occur. As can be seen in Table 2-14, mean levels reported for this industry generally are

less than 15 ppm. IARC (2002) reported concentrations below 0.15 ppm for vulcanization

and extrusion processes involving styrene-butadiene rubber, and exposure to end-users of

styrene-butadiene rubber would likely be even lower. Exposure estimates from the series

of cancer epidemiology studies of styrene-butadiene rubber production workers in North

America (the United States and Canada) (see Section 3.2) are included in Table 2-14. The

highest levels of exposure were reported for recovery operators, unskilled maintenance

workers, and laboratory technicians (Macaluso et al. 2004). Macaluso et al. (1996)

reported that mean styrene exposure levels declined from approximately 2 ppm in the

1940s and 1950s to 0.5 ppm or less in the 1990s; however, workers identified as recovery

operators were frequently exposed to levels of 50 ppm or higher during the 1940s and

1950s.

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In a mortality survey of workers engaged in the production of styrene-based products,

including styrene-butadiene latex, Ott et al. (1980) noted that exposure concentrations

were highest for workers involved in the initial phases of the production process (i.e.,

loading, operating, and cleaning polymerization reactors) (see Section 3.3). Other data

presented by Ott et al. on the styrene monomer and polymer industry are in Table 2-15.

Mean concentrations from personal air samples taken in 1979 at a U.S. styrene-butadiene

rubber production plant were 1.69 ppm for factory service workers and 13.7 ppm for tank

farm workers (IARC 2002). It was noted that mean levels were below 1 ppm for other

departments.

IARC (2002) reported styrene air concentrations of 61 to 146 ppb [0.06 to 0.15 ppm] in

the curing area of the press room of a company that produced car tires. Area air samples

taken in plants producing shoe soles, tire re-treading, and electrical cable insulation

showed styrene levels from 2 to 500 μg/m3 [0.0005 to 0.12 ppm] in vulcanization areas

and from 0 to 20 μg/m3 [0 to 0.005 ppm] in extrusion areas (IARC 2002). Table 2-14

summarizes workplace exposure levels for the styrene-butadiene industry.

Anttinen-Klemetti et al. (2006) assessed exposure to 1,3-butadiene and styrene in three

plants manufacturing styrene-butadiene co-polymers in Finland. A total of 885 air


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samples were collected from the breathing zone of 28 workers over four months. For

styrene, 336 samples (38%) were below the limit of quantitation (0.007 ppm), 548

samples (62%) were between the limit of quantitation and 20 ppm [which is the Finnish

TLV], and one sample (0.1%) exceeded 20 ppm [actual level not reported]. Mean styrene

levels for the three plants were 0.024, 0.07, and 0.188 ppm.

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Table 2-14. Summary of occupational styrene exposure levels in the styrene-butadiene rubber industry

Type of plant (Year measured)

Specific job/process/production

area Mean (range),

ppm Reference (Location)

Synthetic rubber production (1974–1977)

tank farm operator recovery operator finishing operator maintenance, skilled maintenance, unskilled laboratory technician all workers

0.7 (0.14–3.35)a 0.61 (0.12–4.4) 1.0 (0.0–3.7)

0.14 (0.02–0.8) 2.9 (0.11–12.3) 0.6 (0.09–2.86)

0.77 (0–12) (N = 214 total)

Crandall 1981 (NIOSH survey) as reported by Macaluso et al. 2004

Synthetic rubber (estimated exposures for comparison with NIOSH data from 1974–1977)

tank farm operator recovery operator finishing operator maintenance, skilled maintenance, unskilled laboratory technician all workers

1.7 (1–2.4)a 5.5 (2.9–8.5) 1.4 (1.0–1.7) 0.9 (0.6–1.2) 9.4 (5.4–14) 4.6 (3.5–6.9) 1.3 (1.2–1.4)

Macaluso et al. 2004

Styrene-butadiene rubber plant (NR)

across all production areas plant 1 plant 2

0.94 (0.03–6.46) (N = 55) 1.99 (0.05–12.2) (N = 35)

Meinhardt et al. 1982 (USA)

Styrene-butadiene rubber plant (NR)

concentrations across five plants

3.53 (0.29–6.66)b (N = 3,649)

Matanoski et al. 1993 (USA and Canada)

Synthetic rubber industry (NR)

medians across 48–164 specific tasks/plant plant 1 plant 2 plant 4 plant 5 plant 7 plant 8

3.0 2.6 2.7 2.7 3.0 3.0


Styrene-butadiene latex mfg. plants (1965) (1973)

high-exposure jobs during initial phases of production

4–22 (NR)a 3.6–7.3 (NR)

Ott et al. 1980 (USA)


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Type of plant (Year measured)


area Mean (range),

ppm Reference (Location)

Styrene-butadiene rubber plant (1979)

tank farm workers factory service workers other various departments

13.7 (NR) 1.69 (NR)

(< 1.0) (N = 159 total)

IARC 2002 (USA)

Car tire production facility (NR)

curing area of press room NR (0.06–0.15) IARC 2002 (NR)

Shoe sole, tire retreading, and electrical cable insulation plants (NR)

extrusion process vulcanization processes

NR (0–0.005)c NR (0.0005–0.12)c

IARC 2002 (NR)

Styrene-butadiene latex production (1997)

not specified 0.024–0.188 (NR)d (N = 885; 336 samples below LOQ of 0.007

ppm)

Anttinen-Klemetti et al. 2006 (Finland)

LOQ = level of quantitation; NR = not reported. a Numbers in parentheses represent 90% uncertainty interval. b Levels presented are the reported mean value across 5 plants and the range of the mean values for the

individual plants. c Levels presented in μg/m3 in source document. d Range of mean styrene levels for three factories.

2.5.4 The styrene monomer and polymer industry 1 Polystyrene can be manufactured by either a batch polymerization or a continuous

polymerization process (Ott et al. 1980). As reported by Ott et al. (1980), the earliest

manufacturing process was the batch polymerization method at Dow Chemical, but that

process was discontinued by 1951 with the possible exception of some experimental

work on the method. In this method, the benzene-washed polymerization containers were

filled with styrene monomer, sealed, and heated during the polymerization step. A batch

process for suspension polymerization was described in the European Union Risk

Assessment Report for styrene (EU 2002) in which styrene is dispersed in water in the

presence of 0.01% to 0.05% suspending agent and a mixture of organic peroxides or

other polymerization initiator. The reaction mix is heated until polymerization is

substantially complete, and the resulting polymer beads are washed, dried, and pelletized.

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The continuous polymerization process is described briefly in Section 2.2 (Production)

and illustrated in Figure 2-9. As shown below, styrene monomer, which may be mixed


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with a nonpolymerizable volatile diluent, is passed through a series of two or more

reactors with heat exchange zones and agitators (EU 2002). The mixture resulting from

this process contains approximately 85% styrene together with residual monomer and is

transferred to a low-pressure, high-temperature devolatilization tower (labeled as

“Separation Section” below) for removal and recycling of the unreacted monomer and

diluent. The hot polystyrene product is cooled and cut into pellets.

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Figure 2-9. Polymerization of polystyrene by the continuous process Source: Cheresources 2008a.

Styrene exposure levels in the styrene monomer and polymer production industries are

generally much lower than levels in the reinforced-plastics industry, and levels in this

industry have declined over the past several decades. Table 2-15 provides measured air

levels that have been reported in the literature.

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Nicholson et al. (1978) presented data collected in 1974 by NIOSH at a plant that

produced styrene monomer and polystyrene (see Section 3.3). Styrene exposure levels

generally ranged from 5 to 20 ppm in high-exposure areas and were below 1 ppm in low-

exposure areas; however, it was noted that wide excursions from these values occurred at

specific locations.

In the breathing zone of a U.S. plant producing ester-styrene co-polymers, styrene

concentrations ranged from nondetectable (< 1 ppb) to 19.8 ppm with an average of about


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0.6 ppm. It was noted that the highest concentrations occurred during styrene unloading

operations (IARC 2002).

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Some older studies, however, have reported styrene levels in excess of 20 ppm. IARC

(2002) presented data from 8-hour personal air samples taken in 1978, 1979, and 1980 in

U.S. workplaces where polystyrene and acrylonitrile-butadiene-styrene molding was

performed. Styrene levels were 17 to 285 mg/m3 [4.0 to 67 ppm] in 1978, 1.4 to 3.2

mg/m3 [0.33 to 0.75 ppm] in 1979, and below the detection limit of 0.01 mg/m3 [0.002

ppm] in 1980.

Other studies have shown styrene levels that varied mainly with the operations being

performed. Based on five separate industrial hygiene surveys conducted between 1962

and 1976, Ott et al. (1980) reported that TWA exposure levels were below 10 ppm for all

jobs in the styrene monomer production industry, including one where excursions were

measured as high as 50 ppm during the drumming of styrene. In batch polymerization

processes in 1942, styrene levels ranging from 5 to 88 ppm were measured during filling

operations; however, subsequent continuous polymerization processes generally resulted

in personal exposure measurements of 1 ppm or less. Residual styrene monomer

concentrations ranging from less than 1 to 16 ppm have been reported in the vicinity of

polystyrene compounding rolls (used for production of sheets of polystyrene) (Ott et al.

1980).

In a U.S. styrene production and polymerization plant, styrene levels were highest in the

polymerization, manufacturing, and purification areas, where mean exposure levels

ranged from 8 to 35 ppm (IARC 2002). For maintenance, laboratory, and packaging

operations, styrene levels were less than 5 ppm. It was noted that urinary mandelic acid

and blood styrene were not detectable in most samples from workers at the end of a shift.

Thiess and Friedheim (1978) presented styrene air concentrations from periodic air

sampling for 1975 to 1976 in a styrene manufacturing plant and a polystyrene

manufacturing plant, both in Germany (see Table 2-15). As part of the same study,

worker exposures were assessed through air monitoring and assessment of urinary

mandelic acid levels in three plants where polymers containing free styrene were


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converted into finished or semi-finished articles. Of the 83 urine samples, 20 were below

the 10 mg/L detection limit, 4 were greater than 500 mg/L and 5 were greater than 1,000

mg/L [concentrations estimated from a graph]. The authors noted that the facilities with

higher styrene air concentrations had a correspondingly higher number of employees with

high mandelic acid levels.

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Table 2-15. Summary of occupational styrene exposure levels in the styrene monomer and polymer industry in the United States

Type of plant (year monitored)


area Mean (range)

(ppm) Reference (Location)

Styrene monomer and polystyrene plant (1978)

low-exposure areas high-exposure areas

< 1.0a 5.0–20.0a [est. levels]

Nicholson et al. 1978 USA

Ester-styrene co-polymer production (NR)

not specified 0.6 (< 0.001–19.8) (N = 50)

IARC 2002 (USA)

Polystyrene and ABS molding facility (1978) (1979) (1980)

not specified 4–67b,c 0.33–0.75 < 0.002

IARC 2002 (NR)

Styrene monomer production (1962–1976)

not specified < 10 (up to 50) Ott et al. 1980 (NR)

Batch polymerization (1942)

filling operations NR (5–88) Ott et al. 1980 (NR)

Polystyrene production (NR)

compounding and rolling NR (< 1–16) Ott et al. 1980 (NR)

Styrene production and polymerization (NR)

polymerization, manufacturing, and purification areas

maintenance, laboratory, and packaging operations

8–35 (NR) < 5 (NR)

IARC 2002 (USA)

Plant producing styrene monomer and polystyrene (1975–1976)

styrene monomer production

polystyrene production

NR (< 0.01 to 6.84) (N = 60) NR (< 0.01 to 46.92) (N = 70)

Thiess and Friedheim 1978 (Germany)

3 production facilities where styrene polymers are converted into other products (1975–1976)

facility A facility B facility C

< 50–70 (NR) (N = 93) 50–300 (NR) (N = 68) 60–300 (NR) (N = 68)

Thiess and Friedheim 1978 (Germany)

ABS = acrylonitrile-butadiene-styrene; NR = not reported. a Noted as “generally” at these levels although wide excursions were seen. b Presented in units of mg/m3 in source document. c No additional information provided to ascertain if the data were a range of means or the full range of sampling points.


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2.5.5 Other occupational exposures 1 Styrene can occur at low levels in a vast array of industries and occupations. This section

provides information on exposure potential for a number of different industries, focusing

on literature published since the most recent IARC review in 2002. Much lower levels

were reported for occupational exposures outside of the industrial settings presented

above (i.e., levels in the low ppb) and this section again presents air levels in ppb rather

than ppm.

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Due to the potential VOC production from the irradiation of organic matter and the

potential for direct uses and leaks of VOCs during the operation of nuclear power plants,

Hsieh et al. (2006) investigated the composition and concentrations of a number of VOCs

in air-conditioned office space and low-level waste repository sites of three nuclear

power plants in Taiwan in 2000. Depending on the type of building being assessed,

concentrations were presented only for the 10 or 20 most abundant chemicals. The

average styrene level at one low-level waste building was 4.01 ppb by volume (ppbv).

Styrene levels were lower in the administrative buildings of the three power plants (0.21,

0.65, and 1.07 ppbv). While the authors noted that concentrations of aromatics,

chlorofluorocarbons, and chlorinated hydrocarbons were markedly higher in the low-

level waste buildings compared with administrative buildings, there was no indication

that styrene levels were higher.

Lee et al. (2006) used personal and area sampling to investigate levels of styrene and

other pollutants in seven photocopy centers in Taiwan in 2002 and 2003. Concentrations

across the seven facilities ranged from 0.5 to 107 μg/m3 [0.0001 to 0.025 ppb]. Styrene

exposure levels from copied paper have been estimated by the U.S. EPA (EPA 2008b).

Based on the low air concentrations that were estimated [levels not provided], the U.S.

EPA concluded that copied paper does not pose a health risk.

Styrene and 38 other air toxics were measured in worksite air of 11 companies in a

petrochemical complex in Taiwan between 1997 and 1999 (Chan et al. 2006). The mean

concentration was either 9.6 ppb or 13.3 ppb depending on how samples that were below

the limit of detection were treated in the calculation of the mean.


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In a study assessing indoor air quality in printing plants, indoor air was monitored for

several VOCs in seven printing plants of varying sizes in Hong Kong (Leung et al. 2005).

There were a total of 10 sampling points across the seven facilities, and styrene levels

were below detection [0.1 ppb] for four sampling points, while 8-hour TWA values

ranged from 1.4 to 7.1 ppb for the remaining 6 sampling points.

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Using personal monitoring, Thorud et al. (2005) assessed exposure levels of styrene and

several other VOCs during surface coating with acid-curing lacquers and paints in 27

Norwegian woodworking and furniture manufacturing facilities during the late 1990s.

Styrene had a geometric mean level of 0.10 ppm [100 ppb] for nine samples and a range

from 0.01 to 1.47 ppm [10 to 1,470 ppb].

In a study to estimate the level of protection that tollbooths afford workers, Sapkota et al.

(2005) measured styrene air levels in indoor air and outdoor air of a Baltimore Harbor

Tunnel tollbooth in the summer of 2001. For indoor air, the mean styrene concentration

was 0.45 μg/m3 [0.11 ppb] with a range of 0.05 to 1.19 μg/m3 [0.01 to 0.28 ppb], and for

outdoor levels the mean concentration was 0.61 μg/m3 [0.14 ppb] with a range of 0.05 to

1.68 μg/m3 [0.01 to 0.39 ppb].

In an assessment of occupational risks to workers at a hazardous waste incinerator in

Turkey, Bakoğlu et al. (2004) measured levels of numerous pollutants, including styrene,

at two sampling points in the vicinity of the incinerator. The sampling points chosen were

those expected to be where maximum airborne exposures occurred. Single air samples for

each sampling location were taken over 16- to 24-hour periods and contained styrene

levels of 2.98 and 5.7 ppb.

Kim et al. (2003) measured styrene at 3 different locations in a factory producing PVC

film and presented mean levels of 1.8 μg/m3 [0.42 ppb] at two of the sampling locations

and a level of 1.6 μg/m3 [0.38 ppb] at the third location.

In two cooking-ware manufacturing companies where styrene-based resins were used, the

8-hour TWA concentrations of styrene ranged from 0.2 to 81 ppm and two short-duration

samples were 142 and 186 ppm (IARC 2002). Area samples taken at a college sculpture


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class where polyester resins were used contained styrene at concentrations from 0.8 to 1.2

ppm, and two breathing zone air samples contained 2.8 and 3.0 ppm. In a study of

taxidermists who used polyester resins to prepare specimens, air concentrations of styrene

ranged from 21 to 300 mg/m3 [4.9 to 70 ppm]. Firefighters can be exposed to styrene

during firefighting activities: IARC (2002) reported a level of 1.3 ppm during the

knockdown phase of a fire. Styrene air levels exceeding 20 ppm have been reported

during the manufacture of polyester paints, lacquers, and putties, and the application of

polyester putties during cable splicing operations resulted in exposure levels ranging

from 2 to 16 ppm. In a Japanese production plant where buttons were made from

polyester resins, 8-hour TWA levels were 7.1 ppm with a maximum air level of 28 ppm.

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2.6 Biological indices of exposure 11 Direct measures of exposure to styrene in humans have included unmetabolized styrene,

which has been measured in expired air, blood, and urine (Guillemin and Berode 1988),

adipose tissue (Engström 1978), and breast milk (Howard 1989). Although individuals

may differ in their ability to metabolize styrene because of differences in metabolizing

enzymes resulting from genetic polymorphisms (see Section 5.4.5), metabolites of

styrene are widely used as biomarkers of exposure. These metabolites include Phase I

intermediates and their conjugates (Phase II intermediates) of styrene glycol and styrene-

7,8-oxide in blood; and the urinary biomarkers mandelic acid and phenylglyoxylic acid

(IARC 2002), 4-vinylphenol (Manini et al. 2003), and phenylhydroxymercapturic acids

(PHEMAs) from glutathione conjugation of styrene oxides (Ghittori et al. 1997). Finally,

adducts of styrene formed through reaction of styrene-7,8-oxide with albumin,

hemoglobin, and DNA also have been used as biomarkers of exposure. In contrast with

measurements of styrene air concentrations to estimate exposure levels, the use of

biological indices will account for exposures from all exposure routes (i.e., inhalation,

ingestion, and dermal exposure).

The biological indices of exposure for styrene listed here are described briefly below and

the half-lives of styrene-7,8-oxide-DNA adducts are discussed in Section 5.4. In general,

the half-lives in blood for styrene and its metabolites range from less than an hour to

slightly greater than a day [due in part to a biphasic clearance with both a rapid and a


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slow phase for each], while protein adducts have half-lives of one to three months, and

DNA adducts have estimated half-lives ranging from 19 hours for the N7 DNA adducts to

1,320 hours for the O6 DNA adduct. The half-lives of styrene, its metabolites, and

adducts are discussed further in Section 5.4.

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[Since biological measures of exposure are a metric of actual exposure, they often are

considered to be superior to measurements of environmental levels. However, there are a

number of limitations to biological monitoring. Biological monitoring data are difficult to

interpret without information on the kinetics of metabolism and clearance, and the

intensity and duration of personal contact, and therefore, the data are often study-specific

and not generalizable across the body of literature. Also, as noted above, metabolism and

clearance parameters may vary across individuals due to genetic polymorphisms and,

therefore, differences in biological levels across individuals may not reflect accurately

their relative exposure levels. The assessment of metabolites can be complicated by the

fact that often the metabolite being measured is not specific for the agent for which

exposure is being assessed. For example, mandelic acid and phenylglyoxylic acid are not

specific for styrene, but can be metabolically derived from other chemicals. Because

biological measurements account for all exposure routes, exposures outside of the source

of concern can inflate exposure estimates. For styrene, smoking and diet are potential

sources of styrene exposure, and thus, smokers or people who get more styrene through

their diet may appear to have higher occupational exposure levels when compared with

non-smokers. Regardless of these issues, biological monitoring is still an important tool

for assessing exposure, especially when used in concert with environmental levels.]

Styrene levels in blood and levels of the major styrene metabolites mandelic acid and

phenylglyoxylic acid in urine are the most commonly used biological indices of exposure

to styrene (IARC 2002). The American Conference of Governmental Industrial

Hygienists (ACGIH) provides Biological Exposure Indices (BEIs) for mandelic acid plus

phenylglyoxylic acid as the sum of free acid and conjugates in urine, and styrene in

venous blood. These indices are designed to represent the levels of these determinants in

specimens collected from healthy workers exposed to the ACGIH Threshold Limit

Values (TLVs) (see Section 2.7.2 for styrene TLVs). The BEI indicates a marker


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concentration below which nearly all workers should not experience adverse health

effects. The mandelic acid/phenylglyoxylic acid BEI is 400 mg/g creatinine for an end of

shift sample, and the BEI for styrene in venous blood is 0.2 mg/L for an end of shift

sample (ACGIH 2007). Pekari et al. also examined p-hydroxymandelic acid, a minor

metabolite of styrene, as a potential biomarker but concluded that while it might be of

toxicological interest, it is not suitable for monitoring. Storage of samples after collection

might affect urinary mandelic acid and phenylglyoxylic acid levels; therefore, Eitaki et

al. (2008) examined their stability under different storage conditions. They recommended

that urine samples be analyzed on the day of collection; however, if that is not possible,

the urine samples should be stored for no longer than 4 days at a temperature of 4°C or

lower.

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Pekari et al. (1993) evaluated urinary mandelic acid, phenylglyoxylic acid, and styrene as

biomarkers of exposure to styrene and concluded that the sum of the urinary metabolites

(mandelic acid plus phenylglyoxylic acid) in specimens was preferable to the use of

either metabolite alone. The authors noted a close linear relationship between airborne

styrene levels and urinary concentrations of mandelic acid, phenylglyoxylic acid, and

styrene in workers exposed through the lungs, but not in workers exposed mainly through

the skin. For mandelic acid plus phenylglyoxylic acid, correlation coefficients were 0.85

for urinary measurements taken after the work shift and 0.81 for measurements taken the

next morning. Pekari et al. noted that styrene monomer levels in urine were also related

to airborne styrene levels (r = 0.89), and that in principal, styrene levels in urine could be

used to assess exposure. They further noted, however, that the literature is mixed on the

quantitative relationships between styrene in urine and airborne levels.

Similar to the results reported by Pekari et al., Ong et al. (1994) reported good

correlations between styrene levels in air and end-of-shift, creatinine-corrected urinary

levels of mandelic acid (r = 0.83) or phenylglyoxylic acid (r = 0.84), but a better

correlation between styrene air levels and end-of-shift creatinine-corrected levels

mandelic acid and phenylglyoxylic acid combined (r = 0.86). For next-morning urinary

collection, correlation coefficients fell to 0.47 for mandelic acid, 0.61 for phenylglyoxylic

acid, and 0.65 for mandelic acid plus phenylglyoxylic acid. The best correlation,


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however, was between styrene air levels and styrene blood levels (r = 0.87). Styrene in

exhaled breath taken immediately after the work shift also showed good correlation with

airborne styrene levels (r = 0.76), and the authors concluded that styrene in breath could

be a useful indicator for low-level styrene exposure as the method is specific, non-

invasive, and rapid. They further noted for biological monitoring of styrene exposure,

exhaled styrene and blood levels of styrene are preferred by some because mandelic acid

and phenylglyoxylic acid are not specific for styrene, but can be metabolically derived

from other chemicals such as ethylbenzene, phenylglycol, as well as a few common

drugs, and that alcohol consumption can decrease mandelic acid levels. Contributions

from dermal exposure to styrene were not assessed in this study; however, as noted above

in Section 2.5.1, Dutkiewicz and Tyras (1968) noted that urinary mandelic acid does not

provide a reliable index of absorption where there is simultaneous skin and lung

exposure.

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Elia et al. (1980) found an excellent correlation (r = 0.96) between log styrene air

concentrations and log creatinine-corrected urinary mandelic acid, either alone or in

combination with phenylglyoxylic acid. Ikeda et al. (1982) reported that the best

correlation in styrene-exposed workers (r = 0.88) was found between styrene in air and

combined measurements of mandelic acid and phenylglyoxylic acid corrected for

creatinine. Neither Elia et al. nor Ikeda et al. assessed the contribution of dermal styrene

exposure.

Other indicators of exposure that have been used include measurements of styrene in

urine and styrene-7,8-oxide in blood (HSDB 2008a). Mixed data have been reported on

the effectiveness of styrene levels in urine as they relate to exposure levels. Pezzagno et

al. (1985) reported a linear relationship and correlation coefficients for TWA styrene

levels in air and styrene in urine of 0.88 (for exposed workers) and 0.93 (for experimental

volunteers), while Ong et al. (1994) reported a “poor correlation” (r = 0.24) between air

and urinary styrene levels. Tornero-Velez et al. (2001) determined styrene and styrene-

7,8-oxide in human blood and reported detection limits of 2.5 μg/L for styrene and 0.05

μg/L for styrene-7,8-oxide. The authors reported a linear relationship between levels of

styrene in blood and the corresponding air concentrations. Linear regression of logged


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values yielded the following relationship: ln[blood styrene (mg/L)] = -4.35 + 0.97 ln[air

styrene (ppm)] (N = 35, r = 0.89). The authors noted that a styrene exposure level of 50

ppm resulted in a level of 0.57 mg/L styrene in blood at the end of a work shift. Levels of

styrene-7,8-oxide in the blood were significantly correlated with air levels of both styrene

and styrene-7,8-oxide. For styrene-7,8-oxide in blood, linear regression of logged values

yielded the following relationship: ln[blood SO (μg/L)] = -3.23 + 0.415 ln[air styrene

(ppm)] (N = 27, r = 0.73). The contribution of dermal styrene exposure was not assessed

in these studies.

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The conjugated urinary metabolites of 4-vinylphenol, a metabolite of styrene, also have

been studied for use as biomarkers of exposure to styrene. 4-Vinylphenol was found to be

significantly correlated both with airborne styrene (r = 0.607, P = 0.001) and the sum of

mandelic acid and phenylglyoxylic acid (r = 0.903, P = 0.001) in end-of-shift samples

(Manini et al. 2003). Manini et al. reported that while the 4-vinylphenol conjugates

represented only about 0.5% to 1% of the total excretion of styrene metabolites, 4-

vinylphenol is the only styrene metabolite, other than styrene-7,8-oxide, not shared with

ethylbenzene, and is therefore considered to be a highly specific marker for styrene

exposure. Manini et al., however, reported a measurable background level of 4-

vinylphenol for both controls and workers occupationally exposed to styrene; this

background level was highly correlated with smoking, and the authors theorized that it

was possibly also from dietary intake. The authors recommended the use of 4-

vinylphenol as a biomarker for styrene exposure only for ambient concentrations greater

than 1 ppm. The contribution from dermal styrene exposure was not assessed in this

study.

The use of PHEMAs as biomarkers of exposure to styrene has been limited, but Ghittori

et al. (1997) proposed this potential biomarker because the molecules could provide

information on the internal exposure to the R- and S-enantiomers of styrene-7,8-oxide,

which have been reported to differ in their toxicity (see Sections 5.1 and 5.2). The R- and

S-enantiomers of styrene oxide can be conjugated with glutathione to form both R- and S-

diastereoisomers of specific mercapturic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-

cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). Linear


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relationships were found between air concentrations of styrene and concentrations of the

metabolites mandelic acid, phenylglyoxylic acid, and M2 corrected for creatinine, and

urinary styrene not corrected for creatinine. The excretion of mercapturic acids exhibited

a significant correlation with styrene air concentration. The M2 mercapturic acid showed

a better correlation (r = 0.56) with respect to M1-R (r = 0.41) and M1-S (r = 0.36). The

authors noted that the results of this analysis suggest that large inter-individual

differences may occur in the metabolism of styrene to mercapturic acids in humans; the

M1-S to M1-R ratio varying between 7.78 and 41.05. [The contribution of dermal

exposure was not assessed.]

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In a review of mercapturic acids as biomarkers of exposure, Haufroid and Lison (2005)

reported that while excretion of PHEMAs has been shown to be significant, this

correlation is modest when compared with the very good correlation seen with mandelic

acid and phenylglyoxylic acid.

Negri et al. (2006) examined the effects of different storage methods on the stability of

PHEMAs and reported that the metabolites were stable for 1 week at 4ºC and with

repeated freezing and thawing; however, because of an unexplained increase in the

PHEMA levels for samples that were not kept frozen, they recommended that samples

should be frozen as soon as possible after collection and thawed only one time

immediately before the analysis.

The measurement of styrene-induced DNA adducts has been reported (Vodicka et al.

2002a, Vodicka et al. 2003, Vodicka et al. 2002b) (see Section 5.4), and these adducts

have been shown to correlate significantly with measures of styrene exposure, including

styrene in workplace air, styrene in exhaled air, styrene in blood, and urinary mandelic

acid (see Section 5.4.4, DNA adducts). Vodicka et al. (1993) detected 4.7 DNA

adducts/108 nucleotides among 10 hand-lamination workers, and 0.3 adducts/108

nucleotides in 8 controls. Vodicka et al. (2002a, 2002b) reported significant linear

relationships between styrene exposure and DNA adducts of styrene, i.e., the N2- and O6-

guanines, with approximately six-fold higher levels of O6-guanine DNA adducts in hand-

lamination workers as compared with controls. The levels of O6-styrene–guanine DNA


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adducts were significantly correlated with styrene workplace air concentration (r = 0.588,

P < 0.001), duration of employment (r = 0.479, P = 0.002), and exposure coefficient

(workplace air concentration multiplied by years of employment) (r = 0.659, P < 0.001).

Vodicka et al. (2003) reported that O6-styrene-7,8-oxide–guanine DNA adducts were

significantly higher in exposed subjects as compared with controls and were significantly

correlated with workplace styrene air concentration (r = 0.73, P < 0.001) and cumulative

exposure (r = 0.659, P = 0.001). The limit of detection for the DNA adducts was 0.4

adducts per 109 nucleotides [0.04 adducts per 108 nucleotides] (Vodicka et al. 2003).

(Biomarkers of effect (such as DNA repair and toxic endpoints) are discussed in Section

5.)

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Adducts of styrene-7,8-oxide to the N-terminal valine in hemoglobin and to cysteine

residues in albumin and hemoglobin also have been used as biomarkers of exposure to

styrene (Brenner et al. 1991, Christakopoulos et al. 1993, Fustinoni et al. 1998, IARC

2002, Liu et al. 2001, Yeowell-O'Connell et al. 1996, Yuan et al. 2007) that can be used

to estimate exposure over longer periods of one to three months (Fustinoni et al. 1998).

Fustinoni et al. compared levels of styrene-7,8-oxide adducts of albumin and hemoglobin

with the urinary markers of mandelic acid and phenylglyoxylic acid among workers

exposed to styrene in the reinforced-plastics industry and in unexposed subjects. They

found high levels of albumin and hemoglobin adducts of styrene-7,8-oxide in unexposed

controls that were not significantly different from those of the exposed workers. The

authors concluded that cigarette smoking is a source of background levels of styrene-7,8-

oxide–protein adducts and they suggested that hemoglobin adducts of styrene-7,8-oxide

can be detected above background levels only when high-level exposure to styrene exists,

which they considered to be 100 mg/m3 [23.5 ppm]. Vodicka et al. (2003) reported that

N-terminal valine adduct levels were significantly higher in exposed subjects as

compared with controls and were significantly correlated with workplace styrene air

concentration (r = 0.779, P < 0.001) and cumulative styrene exposure (r = 0.657, P =

0.006). Yeowell-O’Connell et al. (1996) found no exposure-related increase in

hemoglobin adducts; however, albumin adducts were found to increase with exposure to

styrene or styrene-7,8-oxide (the latter being more important). High levels were also

found in people without occupational exposure, suggesting to the authors that styrene-


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7,8-oxide is either produced endogenously or exposure was occurring from other sources

(i.e., dietary or other environmental exposures). Significant correlations were found for

the styrene-7,8-oxide–albumin adduct 2-phenylethanol versus styrene air levels (P =

0.017) and styrene-7,8-oxide air levels (P = 0.01). These studies did not evaluate the

contribution of dermal styrene exposure.

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2.7 Regulations and guidelines 6 2.7.1 Regulations 7 Department of Homeland Security 46 CFR 150 and 151 detail procedures for shipping styrene monomer and for shipping

styrene monomer and various styrene co-polymers with incompatible mixtures

Department Of Transportation (DOT) Considered a hazardous material, and special requirements have been set for marking,

labeling, and transporting this material

Environmental Protection Agency (EPA) Clean Air Act 15

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National Emission Standards for Hazardous Air Pollutants: Listed as a hazardous air

pollutant

New Source Performance Standards: Synthetic Organic Chemical Manufacturing

Industry (SOCMI) facilities that meet the definition of a new source and produce

styrene are subject to provisions for the control of VOC emissions

Control of Emissions of Hazardous Air Pollutants from Mobile Sources: Listed as a

mobile source air toxic

Clean Water Act 23

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Styrene has been designated a hazardous substance with a reportable quantity (RQ) of

1,000 lb

Comprehensive Environmental Response, Compensation, and Liability Act 26

27 Reportable quantity (RQ) = 1,000 lb

Emergency Planning and Community Right-To-Know Act 28

29 Toxics Release Inventory: Listed substance subject to reporting requirements


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Safe Drinking Water Act 1

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Maximum contaminant level (MCL) = 0.1 mg/L

Food and Drug Administration (FDA)

Maximum permissible level in bottled water = 0.1 mg/L

The food additive poly (2-vinylpyridine-co-styrene) may be safely used as nutrient

protectant in feed for beef cattle and dairy cattle and replacement dairy heifers with

residual styrene levels not to exceed 200 ppb

Polystyrene basic polymers used as components of articles intended for use in contact

with food shall contain not more than 1 weight percent of total residual styrene

monomer (0.5 weight percent on certain fatty foods)

Rubber-modified polystyrene basic polymers used as components of articles intended for

use in contact with food shall contain not more than 0.5 weight percent of total residual

styrene monomer

Styrene-maleic anhydride co-polymers may be used as articles or as components of

articles intended for use in contact with food provided that conditions detailed in the

regulation are met, including a maximum residual styrene monomer of 0.3% by weight

Styrene-acrylic co-polymers may be used as components of the food-contact surface of

paper and paperboard provided that certain conditions are met, including residual

styrene monomer levels in the polymer not exceeding 0.1% by weight

Occupational Safety and Health Administration (OSHA) Acceptable peak exposure = 600 ppm (5-minute maximum peak in any 3 hours)

Ceiling concentration = 200 ppm

Permissible exposure limit (PEL) = 100 ppm

2.7.2 Guidelines 24 American Conference of Governmental Industrial Hygienists (ACGIH) Threshold limit value – short-term exposure limit (TLV-STEL) = 40 ppm

Threshold limit value – time-weighted average limit (TLV-TWA) = 20 ppm

Biological exposure indices

Mandelic acid plus phenylglyoxylic acid in urine, end of shift = 400 mg/g creatinine


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Styrene in venous blood, end of shift = 0.2 mg/L 1

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National Institute for Occupational Safety and Health (NIOSH) Immediately dangerous to life and health limit (IDLH) = 700 ppm

Short-term exposure limit (STEL) = 100 ppm

Recommended exposure limit (REL) = 50 ppm

2.8 Summary 6 The primary use of styrene is in the manufacture of polystyrene, which is used

extensively in the manufacture of plastic packaging, thermal insulation in building

construction and refrigeration equipment, and disposable cups and containers. Styrene

also is used in styrene-butadiene rubber and other polymers and resins that are used to

manufacture boats, shower stalls, tires, automotive parts, and many other products. U.S.

production of styrene has risen steadily over the past 70 years, with 11.4 billion pounds

produced in 2006 (domestic production capacity for 2006 was estimated at 13.7 billion

pounds). Styrene and styrene metabolites in blood and urine, and styrene-7,8-oxide–DNA

adducts and styrene-7,8-oxide–hemoglobin adducts are generally accepted biological

indices of exposure to styrene. The primary source of exposure to the general public is

inhalation of indoor air; however, exposure can also occur from inhalation of outdoor air,

ingestion of food and water, and potentially from skin contact. Tobacco smoke also can

be a major source of styrene exposure for both active smokers and individuals exposed to

environmental tobacco smoke. Outdoor and indoor air levels (including air levels in most

other occupational settings) are generally below 1 ppb [0.001 ppm]; although higher

levels have been reported. Workers in certain occupations, including the reinforced-

plastics, styrene-butadiene, and styrene monomer and polymer industries, may be

exposed to higher levels of styrene than the general public. Air levels in the reinforced-

plastics industry are generally lower than 100 ppm [although much higher levels have

frequently been measured] while levels in the styrene-butadiene industry and the styrene

monomer and polymer industries have rarely been reported to exceed 20 ppm. Numerous

Federal agencies have established regulations for styrene including the Department of

Homeland Security, DOT, EPA, FDA, and OSHA, and both ACGIH and NIOSH have

established guidelines to limit occupational exposure to styrene.



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3 Human Cancer Studies 1

Three IARC working groups reviewed human studies on the carcinogenicity of styrene in

1979, 1994, and 2002. The 1979 and the 1994 working groups characterized the evidence

available to them at the time on carcinogenicity in humans as “inadequate” (IARC 1979,

1994a). The 2002 working group upgraded the human evidence to “limited” (IARC

2002).

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The 1994 IARC evaluation (IARC 1994a) was based on 19 epidemiologic studies (Bond

et al. 1992, Coggon et al. 1987, Flodin et al. 1986, Frentzel-Beyme et al. 1978, Härkönen

et al. 1984, Hodgson and Jones 1985, Kogevinas et al. 1994a, Kogevinas et al. 1994b,

Kolstad et al. 1994, Matanoski et al. 1993, Matanoski et al. 1990, McMichael et al.

1976a, Meinhardt et al. 1982, Okun et al. 1985, Ott et al. 1980, Santos-Burgoa et al.

1992, Siemiatycki 1991, Wong 1990, Wong et al. 1994) and 3 case reports (Block 1976,

Lemen and Young 1976, Nicholson et al. 1978). The 2002 IARC evaluation added 13

epidemiologic studies not included in the 1994 evaluation (Anttila et al. 1998, Delzell et

al. 2001, Delzell et al. 1996, Dumas et al. 2000, Gérin et al. 1998, Kogevinas et al. 1993,

Kolstad et al. 1995, Kolstad et al. 1993, Loughlin et al. 1999, Macaluso et al. 1996,

McMichael et al. 1976b, Sathiakumar et al. 1998, Sielken and Valdez-Flores 2001).

Cohen et al. (2002)1 included 4 additional epidemiologic studies not reviewed by IARC

(Cantor et al. 1995, Matanoski et al. 1997, Meinhardt et al. 1978, Parent et al. 2000).

This background document reviews the epidemiologic studies (or latest update)

previously reviewed by IARC and Cohen et al. and additional epidemiologic studies

(Coyle et al. 2005, Graff et al. 2005 (also reported in Delzell et al 2006), Guenel et al.

2002, Kolstad et al. 1996, Ruder et al. 2004, Sathiakumar et al. 2005, Scélo et al. 2004,

Seidler et al. 2007), as well as 5 studies that characterized styrene exposure (Crandall

1981, Jensen et al. 1990, Kolstad et al. 2005, Macaluso et al. 2004, Thiess and Friedheim

1978) and were used for several of the epidemiologic studies. One paper published in

2002 (Magnavita et al. 2002) was not included in this review because it was a case report

for a single individual who worked as a boat builder. A population-based study of cancer

1 The expert panel evaluation conducted by the Harvard Center for Risk Analysis and funded by the Styrene Information and Research Center (SIRC).


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among persons with potential occupational exposure to styrene in comparison with other

working men and women, published in 2007 in German (with an English abstract), was

also identified but not reviewed here because only limited details of the study were

reported in the English abstract. (Note that the abstract reported that no significant

increases in all cancers combined or specific cancers (not specified) were observed for

men or women in styrene processing industries, although there were small numbers of

potentially exposed women.)

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In accordance with the IARC evaluation and Cohen et al. (2002), this review is organized

by the three major industrial settings where workers are exposed to styrene – the

reinforced-plastics industry (Section 3.1), the styrene-butadiene rubber industry (Section

3.2), and the styrene monomer and polymer industry (Section 3.3) – because exposure

conditions differ significantly among these industries. A fourth category includes studies

conducted in the general population or other industrial settings (Section 3.4). Section 3.5

describes the available case-control and ecological studies. Section 3.6 discusses

strengths and limitations of the literature, and Section 3.7 summarizes previous

evaluations by IARC (1994a, 2002) and Cohen et al. (2002). Section 3.8 summarizes the

findings for selected cancer sites. Section 3.9 provides an overall summary for this

section.

Tables 3-1 and 3-4 to 3-7 present study characteristics and findings for each individual

study. Tables 3-2 and 3-3 provide specific findings from the largest study of styrene-

butadiene rubber workers (Delzell et al. 2006). In addition, Table 3-8 summarizes the

findings for all cancer sites for all 12 independent cohorts reviewed. Table 3-9 presents

the pooled results for selected cancers (which appear to have the most consistently

increased risks based on the tabulations in Table 3-8 obtained from studies of workers in

the reinforced-plastics industry, and Table 3-10 presents the pooled results for those same

selected cancers among workers in high-styrene–exposure groups (laminators and others)

in the reinforced-plastics industry.


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3.1 The reinforced-plastics industry 1

As noted in Section 2.6.1, the highest occupational exposures to styrene, with respect to

the number of employees and exposure levels, occur in the fabrication of objects such as

boats, car and truck parts, tanks, tubs, and shower stalls from reinforced plastics (IARC

2002). Exposures in this industry have been in the range of several hundred parts per

million in the past, but reported levels have declined over the past several decades.

Workers in the reinforced-plastics industry may also be exposed to other chemicals,

including acetone and other solvents; organic peroxides; cross-linking agents such as

methyl methacrylate; chlorinated hydrocarbons such as dichloromethane; hydroquinone;

oxidation products such as styrene-7,8-oxide; dusts and fibers (such as glass fibers, silica,

asbestos) from filler and reinforcement materials; foaming agents such as isocyanates;

and cobalt salts and amines used as accelerators (EPA 1997b, IARC 2002).

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Cancer mortality or incidence has been studied in the following four populations of

reinforced-plastics workers: (1) in Washington state in the United States (Okun et al.

1985, Ruder et al. 2004), (2) in 30 manufacturing plants in unspecified U.S. locations

(Wong 1990, 1994), (3) in Denmark (Kolstad et al. 1995, Kolstad et al. 1993, Kolstad et

al. 1994, Kolstad et al. 1996) and (4) in Europe (Denmark [a subset of the workers from

the studies by Kolstad et al.], Finland, Italy, Norway, the United Kingdom, and Sweden)

(Kogevinas et al. 1994a, 1993). Results from the U.K. subset of the European population

were also reported separately by Coggon et al. (1987). The Danish and the European

populations were partly overlapping, as 13,682 Danish male workers were included

among the 36,610 male workers in the Danish studies reported by Kolstad et al. (1995,

1994). The two U.S. studies did not overlap. An overview of the individual studies is

presented in Table 3-1.

3.1.1 Washington state 25

Okun et al. (1985) reported on cancer mortality among 5,201 workers (82% men)

employed for at least one day in two reinforced-plastics boat-building facilities in

Washington state between 1959 and 1978. Ruder et al. (2004) extended follow-up

through 1998. Vital status of each subject was determined using data from the Social

Security Administration, Internal Revenue Service, Department of Motor Vehicles, and


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National Death Index. Cause of death was obtained from death certificates. Standardized

mortality ratio (SMR) analyses compared observed deaths classified by the underlying

cause of death with expected numbers computed from state and national rates. Of

workers classified as highly exposed (see below), 74% worked less than 1 year, and 1%

worked more than 10 years. A total of 135,707 person-years were accumulated, and the

average follow-up was 26 years.

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Exposure was assessed using industrial hygiene surveys that classified the jobs and

departments according to the level of styrene exposure. According to this assessment,

2,060 employees (40%) had ever worked in fibrous glass or lamination departments;

these constituted a well-defined high-exposure group. Full-shift average styrene exposure

levels within these departments was 42.5 ppm (range 7.3 to 84.7 ppm) at Plant A and

71.7 ppm at Plant B (range 14.5 to 183 ppm) in 1978 to 1979 (Crandall 1981). A total of

3,141 employees worked with boat assembly, in administration, and in general plant-wide

departments with lower styrene exposure levels; these workers were classified as having

low exposure and were assigned an exposure level of 5 ppm by the authors; no

measurements were reported.

For the total cohort, overall cancer mortality was significantly elevated in comparison

with Washington state reference rates (SMR = 1.17, 95% CI = 1.02 to 1.33, 233 observed

deaths). Statistically significant increases in mortality were also seen for cancer of the

esophagus (SMR = 2.30, 95% CI = 1.19 to 4.02, 12 observed deaths), prostate (SMR =

1.71, 95% CI = 1.09 to 2.54, 24 observed deaths), and other and unspecified sites (ICD-9

codes 194 to 199, SMR = 1.68, 95% CI = 1.01 to 2.62, 19 observed deaths).

Among highly exposed workers, a statistically nonsignificant increase in overall cancer

mortality was observed (SMR = 1.26, 95% CI = 0.96 to 1.63, 58 observed deaths), as

well as statistically nonsignificant increases in mortality due to cancer of the esophagus

(SMR = 1.85, 95% CI = 0.22 to 6.67, 2 observed deaths), stomach (SMR = 1.55, 95% CI

= 0.19 to 5.61, 2 observed deaths), intestine except rectum (SMR = 1.55, 95% CI = 0.50

to 3.63, 5 observed deaths), pancreas (SMR = 1.88, 95% CI = 0.51 to 4.81, 4 observed

deaths), lung (SMR = 1.29, 95% CI = 0.76 to 2.04, 18 observed deaths), ovary (SMR =


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2.32, 95% CI = 0.28 to 8.38, 2 observed deaths), prostate (SMR = 2.06, 95% CI = 0.43 to

6.04, 3 observed deaths), kidney (SMR = 3.60, 95% CI = 0.98 to 9.20, 4 observed

deaths), bladder (SMR = 3.17, 95% CI = 0.38 to 11.5, 2 observed deaths), and brain

(SMR = 1.28, 95% CI = 0.26 to 3.75, 3 observed deaths), and Hodgkin’s disease (SMR =

1.78, 95% CI = 0.05 to 9.89, 1 observed death). Site-specific mortalities were generally

comparable for the low-exposure group except for cancers of the urinary organs, which

were higher in the high-exposure group.

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In general, gender-specific SMRs were not calculated; however, the authors noted that

there was a statistically nonsignificant increase in lung cancer mortality among white

females (SMR = 1.82, 95% CI = 0.78 to 3.59, 8 observed deaths), and among white

females with high exposure to styrene (SMR = 2.11, 95% CI = 0.77 to 4.60, 6 observed

deaths).

Among workers employed for at least one year (N = 1,678; 580 high exposure, and 1,098

low exposure), statistically nonsignificant increases in SMRs were observed in high-

exposure departments compared with low-exposure departments, for cancer of the

esophagus, intestine (not including rectum), kidney, and bladder. [This analysis was

limited by small numbers of expected and observed cancer deaths in the high-exposure

subcohort of workers employed for more than one year (all cancer deaths, 20 observed

and ~22 expected deaths).] A statistically nonsignificant increase in overall mortality was

found among workers employed for less than 1 year (short-term workers). The authors

also stated that for urinary cancer, there was a trend towards increasing mortality with

increasing duration of employment in the high-exposure departments, and also with

increasing levels (terciles) of cumulative exposure.

The authors stated that the study was limited by lack of information on lifestyle choices,

previous or subsequent employment, exposure to other occupational agents, and job

information after 1978. Cumulative exposure estimates were not job specific and did not

include any exposures between 1978 and when the plant closed (1989 for Plant B and

1993 for Plant A). They noted that the lack of job information after 1978 meant that

cumulative exposure and duration of exposure are underestimates and would bias results


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towards the null hypothesis. The authors also stated that the work-history records did not

include specific job titles and that the exposures varied widely with the high-exposure

departments.

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3.1.2 United Kingdom 4

Coggon et al. (1987) studied 7,949 workers (6,638 men and 1,311 women) at eight

reinforced-plastics companies in the United Kingdom. All employees, regardless of

duration of exposure, were included between 1947 and 1984 (inclusion periods differed

among the companies) and followed through 1984. Follow-up was incomplete for 3%.

For one company, sufficient employment data were identified from personnel records for

only 61.9% of the employees, and results for these workers were presented separately

from the detailed analyses. Vital status was traced through the National Health Service

Cancer Register, and National Insurance Index. Cause of death was obtained from death

certificates. Mortality was compared with expected values computed from national

mortality rates. Gender-specific SMRs were not calculated. Durations of employment

were less than 1 year for 51% of the workers and 10 years or more for 8%. From

personnel records, workers were classified into four categories: hand laminators (high

exposure) (44%), regular bystander exposure (7%), occasional bystander exposure (17%),

or background exposure (32%). The authors estimated that hand laminators were exposed

to styrene at levels of 40 to 100 ppm 8-hour TWA, based on measurements conducted at

the companies since 1975; however, no styrene exposure measurements were presented.

Among all workers at the seven companies with almost complete data, a statistically

significant decrease in the SMR for overall cancer mortality was observed (SMR = 0.80

(95% CI = 0.69 to 0.93, 167 observed deaths). The SMR did not differ statistically from

unity for any specific cancer, but statistically nonsignificant increases (> 10%) in SMRs

were observed for larynx (SMR = 1.16, 95% CI = 0.14 to 4.18, 2 observed deaths), lung

(SMR = 1.12, 95% CI = 0.89 to 1.39, 83 observed deaths), melanoma (SMR = 1.19, 95%

CI = 0.14 to 4.30, 2 observed deaths), non-melanoma skin cancer (SMR = 3.57, 95% CI

= 0.43 to 12.90, 2 observed deaths), and cancer of the ovary (SMR = 1.49, 95% CI = 0.41

to 3.81, 4 observed deaths). Lung cancer mortality was highest in individuals with

moderate and high exposure, and individuals exposed from 1 to 9 years, but the exposure-


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response relationship was not consistent. Mortality of other cancers was not consistently

related to first year of exposure, duration of exposure, or latency for any type of cancer;

however, the numbers of deaths were small. Among hand laminators (the well-defined

high-exposure category), mortality was increased (statistically nonsignificant) for cancer

of the large intestine, lung, cervix, ovary, and prostate. Kogevinas et al. included this

population in the European study, with follow-up extended through 1990 (Kogevinas et

al. 1994a, 1993).

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The authors stated that the study had limited power to detect cancers with long latency

(only 5 cancer deaths were expected among workers exposed at least 12 months with a

latency period of 20 years).

3.1.3 United States 11

Wong et al. reported on cancer mortality among 15,826 workers (75.6% men) at 30 U.S.

reinforced-plastics plants between 1948 and 1977 (Wong 1990) and later extended

follow-up through 1989 (Wong et al. 1994). Employees who had worked for at least 6

months in an area with potential exposure to styrene were included in the study.

However, not all workers actually worked in activities that entailed direct and significant

exposure to styrene (Wong 1990). The duration of employment was less than 1 year for

24% of the workers and 5 years or more for 27%. Vital status information was obtained

from the participating plants, the Social Security Administration, the National Death

Index, the National Center for Health Statistics, and a commercial retail credit bureau.

Cause of death was determined from death certificates. Vital status was unknown for 547

workers at the end of follow-up, and death certificates were not identified for 42; loss to

follow-up was thus 3.7%. Standard SMR analyses were conducted, based on expected

values computed from national death rates for whites (no information on race was

available for the study population), as well as internal Cox regression analyses. Gender-

specific risk estimates were not calculated. Odds ratios (ORs) for respiratory cancer

mortality were computed by the Mantel-Haenszel procedure among 40 cases and 102

controls nested within the study population. This analysis included information on

smoking obtained from 78% of cases and 61% of controls (Wong 1990).


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A job exposure matrix was constructed for each plant from styrene measurements and

process descriptions collected circa 1980. Only 43% of the total study population had

direct exposure to styrene, according to information obtained for the 102 controls

included in the nested case-control study (Wong 1990). The estimated styrene TWA

values by job were 60 ppm (5 to 120 ppm) for spray and hand lay-up; 20 to 45 ppm for

sheet molding, gel coating and winding; and 2 to 7 ppm for office, injection molding,

field service, finish and assembly, store and ship, and preform production (Wong et al.

1994). The worker population was then classified into six processing categories based on

exposures to styrene and other substances: open-mold processing, mixing and closed-

mold processing, finish and assembly, plant office and support, maintenance and

preparation, and supervisors and professionals. Cumulative exposure to styrene was

estimated, taking account of job changes and duration of employment (Wong et al. 1994).

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For the total study population, the SMR for all cancer was 1.16 (95% CI = 1.05 to 1.27,

425 observed deaths) (Wong et al. 1994). Statistically significantly increased SMR

values were also seen for cancer of the esophagus (SMR = 1.92, 95% CI = 1.05 to 3.22,

14 observed deaths), lung (SMR = 1.41, 95% CI = 1.20 to 1.64, 162 observed deaths),

cervix (SMR = 2.84, 95% CI = 1.36 to 5.21, 10 observed deaths), and other female

genital organs (SMR = 2.02, 95% CI = 1.07 to 3.45, 13 observed deaths). Decreased risks

were seen for all lymphatic and hematopoietic malignancies (SMR = 0.82, 95% CI = 0.56

to 1.17, 31 observed deaths) and among the subcategories (lymphosarcoma, Hodgkin’s

disease, leukemia, or cancer of all other lymphopoietic tissue). A statistically

nonsignificant increase in mortality from pancreatic cancer was observed (SMR = 1.13,

95% CI = 0.68 to 1.77, 19 observed deaths), while mortality from laryngeal cancer was as

expected.

The category with the expected highest styrene exposure levels was workers employed in

open-mold processing. Among those working for more than two years in this category

[the only results presented for this category], statistically nonsignificant increases in

mortality were seen for cancer of the esophagus, stomach, uterus, cervix, kidney,

lymphosarcoma, and all other lymphopoietic tissue. Mortality for pancreatic cancer, lung

cancer, Hodgkin’s disease, and leukemia was decreased. These findings were based on


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few observed cases. Among workers with > 2 years employment in the other 5 work

categories, statistically significant increases in mortality were observed for cancer of the

biliary tract and liver among office and support workers (SMR = 4.56, P < 0.05, 4

observed deaths), and for bronchus, trachea, and lung cancer among maintenance and

support workers (SMR = 1.49, P < 0.05, 30 observed deaths).

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Statistically nonsignificant increases in SMRs [CIs not reported] were observed for all

long-term workers (≥ 10 years) for cancer of the esophagus (SMR = 2.13, 4 observed

deaths), and cervix (SMR = 4.04, 2 observed deaths), lymphosarcoma (SMR = 1.86, 2

deaths), and cancer of all other lymphopoietic tissue (SMR = 1.32, 4 observed deaths), as

well as for several other cancers, but this analysis did not take exposure level into

consideration. Cox regression analyses (internal analysis) of cumulative styrene exposure

or duration of styrene exposure showed no indications of exposure-response relationships

for cancer of the esophagus, lung, uterus, other female genital organs, kidney, or all

lymphopoietic tissue, non-Hodgkin’s lymphoma (NHL), multiple myeloma, or leukemia

(Wong et al. 1994). In addition, no trends were seen in SMR analyses of duration of

employment, duration of styrene exposure, or cumulative styrene exposure. However,

lung cancer mortality increased with latency; statistically significantly increased SMRs

were seen for workers with latencies of 10 to 19 years or at least 20 years. The nested

case-control study (15 cases and 44 matched deceased controls) showed no increased risk

of lung cancer among workers with direct exposure to styrene (Wong 1990).

3.1.4 Denmark 21

Kolstad et al. (1995, 1994) studied the incidence of cancer among 36,610 male workers

at 386 Danish reinforced-plastics companies and a reference population of 14,293 male

industrial workers at 84 companies with no styrene exposure. The method of exposure

classification of workers in the Kolstad cohort was based on data obtained from two

independent dealers (who identified the companies from a list of 552 likely producers of

reinforced plastics) rather than information obtained from employers. The decision to do

so was based partly on indications that the employers’ exposure assessments were not

independent of health outcomes for some companies. The two independent dealers agreed

on all but 4 of 328 companies that they could both classify (kappa = 0.94) (80 companies


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remained unknown to one or both of the dealers). There was agreement with the

employers in 281 of 302 companies known to both. In addition, there was good

agreement between employers and dealers on the proportion of all employees in a

company engaged in the production of reinforced plastics. For 287 companies (12,862

workers), the estimate was 50% to 100%, and for 99 companies (23,748 workers), the

estimate was 1% to 49%. [Nevertheless, the posthoc decision to rely solely on the

dealers’ estimates of exposure, together with a lack of exposure measurements (except in

a small sample of the companies included in the study in a separate survey) represents a

methodological limitation of this study.]

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Female workers were included in an early overview of this population but were omitted

from subsequent studies because the majority were not involved in the production of

reinforced plastics (Kolstad et al. 1993). The population was followed from 1970 to 1989

(Kolstad et al. 1994) or 1990 (Kolstad et al. 1995), and loss to follow-up was 2%. Cancer

cases were identified in the national cancer registry, and standardized incidence ratios

(SIRs) were computed from the national reference rates. In internal analyses, Poisson

regression models were used to estimate incidence rate ratios (IRRs). A total of 618,900

person-years were accumulated, and the average follow-up was 10.9 years. No

information was available on individual indicators of exposure such as task or job title,

but time and duration of employment were reported in a national pension scheme for the

period 1964 to 1988. The duration of employment was less than 1 year for 60% of the

workers and 10 years or more for 3%.

Measurements of styrene exposure levels in the industry were available back to the early

1960s, based on 2,473 personal air samples collected by the work inspection service.

About 90% of the samples were taken during lamination procedures (Jensen et al. 1990).

The mean period-specific styrene exposure levels were 180 ppm (1964 to 70), 88 ppm

(1971 to 75), and 43 ppm (1976 to 88), and an estimated 43% of the study population

were laminators (Kolstad et al. 1994).

No SIR for overall cancer incidence was reported for all workers (at the 386 reinforced

plastic companies), but the incidence of all solid cancers was as expected (SIR = 0.99,


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95% CI = 0.93 to 1.05, 1,134 observed cases) (Kolstad et al. 1995), and a statistically

nonsignificant increase in the incidence of all lymphohematopoietic malignancies was

observed (SIR = 1.20, 95% CI = 0.98 to 1.44, 112 observed cases) (Kolstad et al. 1994).

Analyses of specific cancers revealed no statistically significantly increased SIRs

(Kolstad et al. 1995, 1994). Incidences were nonsignificantly increased for cancer of the

pancreas (SIR = 1.20, 95% CI = 0.86 to 1.63, 41 observed cases), nasal cavities (SIR =

1.84, 95% CI = 0.74 to 3.80, 7 observed cases), lung (SIR = 1.12, 95% CI = 0.98 to 1.26,

248 observed cases), pleura (SIR = 1.78, 95% CI = 0.85 to 3.28, 10 observed cases),

external male genital organs (SIR = 1.60, 95% CI = 0.64 to 3.30, 7 observed cases), and

bladder (SIR = 1.16, 95% CI = 0.96 to 1.39, 117 observed cases) (Kolstad et al. 1995),

NHL (SIR = 1.33, 95% CI = 0.96 to 1.80, 42 observed cases), and leukemia (SIR = 1.22,

95% CI = 0.88 to 1.65, 42 observed cases) (Kolstad et al. 1994).

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The risks of cancers with elevated SIRs were further evaluated by an internal analysis

using workers not exposed to styrene as controls. The category with the highest potential

styrene exposure was workers at companies with 50% to 100% laminators. Among these

workers, there was a statistically significant excess of pancreatic cancer (IRR = 2.2, 95%

CI = 1.1 to 4.5, 17 observed cases) (Kolstad et al. 1995). The risk was higher in long-

term workers (≥ 1 year) than in short-term workers and among workers with earlier first

years of employment (1970 or before) than later; however, latency had no influence on

IRR values. The risk of lung cancer was not increased (IRR = 1.0, 95% CI = 0.7 to 1.3,

72 cases) among workers with higher exposure potential, and lower risk was seen in

long-term workers. Analyses by first year of employment, length of employment, and

latency revealed no consistent findings for cancer of the nasal cavities, pleura, external

male genital organs, or urinary bladder, NHL or the other lymphohematopoietic

malignancies except for leukemia (Kolstad et al. 1994, 1995).

The risk of leukemia was non-significantly increased with the probability of exposure to

styrene (SIR = 1.38, 95% CI = 0.75 to 2.32, 14 observed cases for high probable

exposure) and significantly increased among workers with earlier date of first

employment (SIR = 1.54, 95% CI = 1.04 to 2.19, 30 observed cases for employment

during the 1960’s), and with latency (SIR = 1.57, 95% CI = 1.07 to 2.22, 32 observed


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cases for at least 10 years since first employment). However, no excess of leukemia was

apparent among those employed for 1 year or more (Kolstad et al. 1994).

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In a case-control study nested within the cohort, Kolstad et al. (1996) studied the risk of

myeloid leukemia with clonal chromosome aberrations, based on 12 cases (out of 34

cases of myeloid leukemia) and 57 non-exposed controls selected from the study

population. Exposure classification relied on company-level assessments, as in the

previous studies (Kolstad et al. 1995, Kolstad et al. 1994). A statistically nonsignificant

odds ratio of 2.5 (95% CI = 0.2 to 25.0) was observed for reinforced-plastics workers

(i.e., for any styrene exposure); the risk was higher among workers employed for less

than 1 year (OR = 5.9, 95% CI = 0.5 to 74.3, 8 exposed cases) than workers employed

longer than 1 year (OR = 1.1, 95% CI = 0.1 to 15.3, 3 exposed cases. [However, the risk

estimates were imprecise.]

3.1.5 Denmark, Finland, Norway, Italy, Sweden, and the United Kingdom. 13 Kogevinas et al. (1994a, 1993) reported on cancer mortality among 40,688 employees at

660 companies in Denmark (15,867), Finland (2,085), Norway (2,035), Italy (7,256),

Sweden (3,667), and the United Kingdom (9,778). Cancer mortality among 7,971 of the

U.K. workers was previously reported by Coggon et al. (1987) but with a shorter follow-

up. The international study included the male (13,682) workers that were also included in

the cancer incidence studies of the Danish population (Kolstad et al. 1995, Kolstad et al.

1994) and the the female (2,185) workers that were not included in those studies from

287 Danish plants where reinforced plastics were the main products produced. The

follow-up periods started between 1945 (United Kingdom) and 1970 (Denmark) and

ended between 1987 (Sweden) and 1991 (Norway). The duration of employment was less

than 2 years for 60% of the workers and 10 years or more for 9%. Loss to follow-up was

1.4%, and the average follow-up was 13 years. In SMR analyses, cancer mortality was

compared with expected mortality computed from national reference rates. In internal

analyses, Poisson regression models were used to compare exposure-specific cancer rates

(rate ratios) and conduct trend analyses.

From job titles recorded on individual payroll records, the population was categorized as

laminators (N = 10,629), workers with unspecified tasks (19,408), other exposed workers


9/29/08 97

with bystander exposure (5,406), workers not exposed to styrene (4,044), and workers

with unknown job titles (1,201). The 15,867 Danish workers were categorized as workers

with unspecified tasks, because no job titles were available. An exposure matrix was

constructed from 16,500 personal styrene measurements obtained between 1955 and

1990 and from 18,500 measurements of styrene metabolites in urine conducted in the

1980s. Styrene exposure levels averaged across country, period, and job were linked with

the individual workers, and cumulative styrene exposure was estimated from additional

information on duration of exposure. All Danish workers and other workers classified as

having unspecified tasks were assigned a styrene exposure level that was the average

value for the calendar period and branch of the industry (boats vs. other). Styrene

exposure levels recorded among laminators declined from about 200 ppm before 1965 to

below 80 ppm in the 1980s.

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30

Among the total study population, overall cancer mortality was statistically significantly

lower than in the European reference population (SMR = 0.87, 95% CI = 0.81 to 0.94,

686 observed deaths). This was also the case for cancer of the buccal cavity and pharynx

(SMR = 0.33, 95% CI = 0.11 to 0.77, 5 observed deaths), rectum (SMR = 0.62, 95% CI =

0.38 to 0.95, 21 observed deaths), breast (SMR = 0.52, 95% CI = 0.28 to 0.89, 13

observed deaths), and brain (SMR = 0.62, 95% CI = 0.37 to 0.98, 18 observed deaths).

No site-specific SMR values were statistically significantly above unity, but excesses

were seen for small intestine (SMR = 1.50, 95% CI = 0.31 to 4.38, 3 observed deaths),

larynx (SMR = 1.11, 95% CI = 0.53 to 2.05, 10 observed deaths), ovary (SMR = 1.40,

95% CI = 0.70 to 2.51, 11 observed deaths) and myeloid leukemia (SMR = 1.10, 95% CI

= 0.63 to 1.79, 16 observed deaths) (Kogevinas et al. 1994a).

The worker category with the best-documented high-level styrene exposure was

laminators. Laminators showed no statistically significant elevated mortality from cancer

at any site, but statistically nonsignificant increases in mortality were observed for cancer

of the esophagus (SMR = 1.82, 95% CI = 0.87 to 3.34, 10 observed deaths), small

intestine (SMR = 2.27, 95% CI = 0.06 to 12.66, 1 death), pancreas (SMR = 1.48, 95% CI

= 0.76 to 2.58, 12 observed deaths), larynx (SMR = 1.55, 95% CI = 0.32 to 4.52, 3

deaths), ovary (SMR = 2.61, 95% CI = 0.71 to 6.69, 4 observed deaths), and thyroid


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(SMR = 2.27, 95% CI = 0.06 to 12.66, 1 observed death), NHL (SMR = 1.40, 95% CI =

0.56 to 2.88, 7 observed deaths), and Hodgkin’s disease (SMR = 1.33, 95% CI = 0.27 to

3.88, 3 observed deaths) (Kogevinas et al. 1994a).

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19

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27

Among workers classified as having unspecified tasks, overall cancer mortality was

greater than expected (SMR = 1.06, 95% CI = 1.00 to 1.12, 1,167 observed deaths), but

no statistically significant increases were seen for any of the specified cancers

(Kogevinas et al. 1994a).

Lower SMRs were observed for long-term workers (≥ 2 years) for all

lymphohematopoietic malignancies, Hodgkin’s disease, multiple myeloma, and

leukemia, but not for NHL (< 2 years: SMR = 0.60, 95% CI = 0.19 to 1.40, 5 observed

deaths; ≥ 2 years: 1.05, 95% CI = 0.42 to 2.17, 7 observed deaths). More than 20 years

after first exposure, a statistically nonsignificant increase in mortality for NHL in long-

term workers was observed (SMR = 2.21, 95% CI = 0.45 to 6.45, 3 observed deaths).

Among workers exposed for at least 2 years, mortality was non-significantly elevated for

all lymphohematopoietic malignancies (SMR = 1.73, 95% CI = 0.70 to 3.57, 7 observed

deaths) and leukemia (SMR = 1.94, 95% CI = 0.40 to 5.66, 3 deaths) in workers with at

least 20 years latency, but not in those with latency of 10 to 19 years (Kogevinas et al.

1994a).

In internal analyses, the relative risk increased with increasing latency for all

lymphohematopoietic malignancies (P = 0.012), leukemia (P = 0.094), and malignant

lymphoma (NHL and Hodgkin’s disease, P = 0.072). Similarly, the rate ratio increased

with increasing average styrene exposure for all lymphohematopoietic cancers (P for

linear trend = 0.019) and for malignant lymphoma (P = 0.052), though not for leukemia

(P = 0.47). A trend for increased risk of pancreatic cancer with increasing cumulative

styrene exposure was of borderline significance (P = 0.07), but no such trend was seen

for all cancer, cancer of esophagus, lung, or kidney, all lymphohematopoietic

malignancies, leukemia, or malignant lymphoma (Kogevinas et al. 1994a).


Table 3-1. Epidemiologic studies of cancer risk following styrene exposure in the reinforced-plastics industry, 1985–2004 (results of the most recent follow-upa)

Study

Study design & follow-up

Study population and methods

Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments

Okun et al. 1985 Ruder et al. 2004

U.S.

historical cohort 1959–98 avg. 26 yr 135,707 person-years

5,201 workers (4,520 men, 681 women) at 2 reinforced-plastics plants employed ≥ 1 day, 1959–78 74% worked < 1 yr and 1% > 10 yr SMRs based on state and national rates; effects reported here based on state rates

Industry hygiene surveys Workers employed in fibrous glass or lamination departments (40% of population) classified as highly exposed. Exposure Levels High-exposure workers Average TWA (ppm) 1978–79 (Crandall 1981) Plant A: 42.5 Plant B: 71.7 Low-exposure workers 5 ppm (assigned not measured)

Total cohort Cancers with excess mortality (significant) overall 1.17 (1.02–1.33), 233 esophagus 2.30 (1.19–4.02), 12 prostate 1.71 (1.09–2.54), 24 unspecified 1.68 (1.01–2.62), 19

High-exposure workers Cancers with non-significant excess mortality overall 1.26 (0.96–1.63), 58 esophagus 1.85 (0.22–6.67), 2 stomach 1.55 (0.19–5.61), 2 intestinec 1.55 (0.50–3.63), 5 pancreas 1.88 (0.51–4.81), 4 lung 1.29 (0.76–2.04), 18 ovary 2.32 (0.28–8.38), 2 prostate 2.06 (0.43–6.04), 3 kidney 3.60 (0.98–9.20), 4 bladder 3.17 (0.38–11.5), 2 brain 1.28 (0.26–3.75), 3 Hodgkin’s 1.78 (0.05–9.89), 1 SMR for lung cancer no longer elevated in high exposed workers exposed for>1 yr Statistically significant elevated SMRs were also observed for cancers of the esophagus, prostate and unspecified sites in the low exposed workers Urinary tract cancer Trend towards increasing SMR with increasing

Job information not available after 1978 No information on lifestyle factors or other environmental exposures

9/29/08 99


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments duration of exposure, and level (tercile) of exposure

Coggon et al. 1987

U.K.

historical cohort 1947–84

7,949 (6,638 men and 1,311 women) workers employed in 8 reinforced-plastics companies 1947–84 (differing periods for each company) 51% worked < 1 yr and 8% worked ≥ 10 yr 979 workers excluded from exposure-related analyses SMRs based on national rates

From personnel records, workers were classified as hand laminators (high exposure) (44%), regular bystander exposure (7%), occasional bystander exposure (17%), or background exposure (32%) Authors estimated TWA styrene exposure levels for hand laminators at 40–100 ppm

Total cohort (7 of the 8 companies)d all cancer 0.80 (0.69–0.93), 167

Cancers with non-significant excess mortality (> 10%) larynx 1.16 (0.14–4.18), 2 lung 1.12 (0.89–1.39), 83 melanoma 1.19 (0.14-4.30), 2 non-melanoma

skin cancer 3.57 (0.43–12.9), 2 ovary 1.49 (0.41–3.81), 4 Lung cancer mortality was highest among individuals with moderate and high exposure, and individuals exposed from 1 to 9 yr, but the exposure response relationship was not consistent. Mortality of other cancers was not consistently related to first year of exposure, duration of exposure, or latency for any type of cancer

Laminators Cancers with non-significant excess mortality large intestine 1.40, 5 lung 1.20, 25 cervix 1.96, 1 ovary 2.82, 2 prostate 1.20, 2

Low statistical power No data on smoking Included in the European multinational study by Kogevinas et al. (1994a)

Wong 1990

U.S.

historical cohort 1948–77 nested case-control study

Historical cohort 15,908 workers employed ≥ 6 mo in 30 reinforced-plastics companies in work

Cohort study Work histories were obtained from employment records. An industrial hygiene survey, which

Cohort study: Repiratory cancer total cohort 1.16, 34 No clear trend of increasing mortality with increasing duration of employment or increase in exposure (potential maximum TWA or average

Young cohort; 46% worked for less than 2 years Four cases identified in the


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments of respiratory cancer

areas with exposure to styrene 1948–77; 499 deaths were observed SMRs based on national rates

Nested case-control study Cases: 40 respiratory cancer deaths; 44 deaths occurred in the cohort (including deaths after the observation period), but eligible controls could not be found for 4 deaths Controls: 102 matched controls; deceased members of cohort, maximum of 3 per case, matched for plant, age at death (within 5 yr), year of death (within 5 yr), sex, and race ORs calculated by Mantel-Haenszel, and logistic regression methods

contained current or past TWA and peak range exposure values, was used to consolidate record job titles (from personnel records) into study job titles. This information was incorporated into a job dictionary and used to classify individuals into exposure groups. Case-control study More detailed work history (than for the cohort analysis), exposure of each job segment, exposures from employment outside the plastics industry, and smoking history were obtained from employment records, medical and insurance records, and some interviews with next of kin or co-workers.

TWA) Subgroups with excess respiratory cancers Respiratory cancer mortality increased non-significantly with increasing latency. Among workers with ≥ 20 years latency; significant SMRs for lung cancer were observed in women (total across all durations of exposure) and men who worked 2–5 years. Higher SMRs were observed in workers in hot process than cold process

(See later update [Wong et al. 1994] for findings on cancer at other sites)

Case-control study of respiratory cancer Mantel-Haenszel OR, cases/controls, P styrene (direct) exposure 0.63, 15/44, 0.29 Logistic regression Only smoking showed an association in a multivariate analysis that included direct exposure to styrene, duration of exposure, type of exposure (hot or cold process), smoking, and interaction terms.

cohort were not used in the case-control analysis Potential exposure to styrene is higher for cold process than hot process

Wong et al. historical cohort

Same cohort as Wong 1990, except 15,826

Company-specific JEMs constructed from

Total cohort (1,628 deaths) Small proportion of the total study


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments 1994

U.S.

1948–89 avg. 19.5 yr 307,972 person-years

(75.6 % men) (identified 30 with duplicate records and 52 who had worked less than 6 months) 24% worked < 1 yr and 27% ≥ 5 yr SMRs based on national rates Cox proportional hazard models including age, sex, exposure duration, and cumulative exposure used for internal analysis

measurements and production characteristics, linked with workers by job and department reported on employment records The worker population was classified into six process categories based on exposures to styrene and other substances: open-mold processing, mixing and closed-mold processing, finishing operations, plant supports, maintenance and preparation, and supervisory and professional Approximately 12% of the workers were engaged in open-mold processing, with estimated average TWAs of 20–60 ppm Other worker categories exposed at average ≤ 5 ppm TWA

Cancers with significant excess mortality all cancer 1.16 (1.05–1.27), 425 esophagus 1.92 (1.05–3.22), 14 cervix 2.84 (1.36–5.21), 10 other female genital organs 2.02 (1.07–3.45), 13 lung 1.41 (1.20–1.64), 162 No exposure-response relationship (cumulative exposure or duration) observed for any cancer

Lung cancer Latency (years since first employment) < 10 1.07, 23, P > 0.05 10–19 1.46, 70, P < 0.01 ≥ 20 1.51, 69, P < 0.01

Internal analysis (stepwise regression and multivariate); entire cohort Cumulative exposure or duration of exposure was not associated with increased mortality from cancers of the esophagus, lung, female genital organs uterus, kidney, lymphohematopoietic tissue, NHL, multiple myeloma, or leukemia

High-exposure workers (open mold processing category) with > 2 years of exposure A statistically nonsignificant excess in mortality was observed for cancer of the esophagus, stomach, uterus, cervix, kidney, lymphosarcoma, and cancer of all other lymphopoietic tissues. SMRs based on small numbers of observed deaths

population was exposed to high styrene levels Cox regression models of cumulative styrene exposure may have been over-controlled by the inclusion of duration of employment SMRs for high-exposure workers based on small numbers of observed and expected deaths

Kolstad et al. historical 36,610 workers at 386 No data on individual Total cohort (Kolstad et al. 1994 LH, 1995, solid Imprecise


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments 1995, Kolstad et al. 1993, Kolstad et al. 1994

Denmark

cohort 1970–89 avg. 10.9 yr 618,900 person-years

reinforced-plastics companies employed > 1 day 1964–88 60% worked < 1 yr, 3% worked ≥ 10 yr SIRs based on national rates 14,293 workers in 84 companies not producing reinforced plastics were used as controls in the internal analysis Incidence RRs were calculated in the internal analysis using Poisson regression models that included the following variables: exposure probability (none, low, high), age, year of first employment, duration of employment and time since first employment

exposure or job titles was available; information on duration of employment was obtained from a national pension fund Classification of exposure was based on percent of workforce producing reinforced plastics Probable high exposure: ≥ 50% of the workforce producing plastics: 12,862 employees Possible low exposure: < 50% of the workforce producing plastics: 23,748 employees An estimated 43% of the study population were laminators Historical personal air samples (N = 2,473) showed average styrene levels declining from 180 ppm (1964–70) to 43 ppm (1976–88) (Jensen et al. 1990)

tumors) all solid cancers 0.99 (0.93–1.05), 1,134

Cancers with non-significantly elevated SIRs pancreas 1.20 (0.86–1.63), 41 nasal cavities 1.84 (0.74–3.80), 7 lung 1.12 (0.98–1.26), 248 pleura 1.78 (0.85–3.28), 10 external male genital organs 1.60 (0.64–3.30), 7 bladder 1.16 (0.96–1.39), 117 LH 1.20 (0.98–1.44), 112 NHL 1.33 (0.96–1.80), 42 leukemia 1.22 (0.88–1.65), 42

Leukemia, by employment variables (Kolstad et al. 1994) Probable exposure high 1.38 (0.75–2.32), 14 low 1.15 (0.77–1.67), 28

Year of first employment 1964–70 1.54 (1.04–2.19), 30 1971–75 1.00 (0.46–1.90), 9 1976–88 0.51 (0.11–1.50), 3

Years since first employment < 10 0.71 (0.34–1.31), 10 ≥ 10 1.57 (1.07–2.22), 32

Years of employment (≥ 10 years latency) < 1 2.34 (1.43–3.61), 20 ≥ 1 1.01 (0.52–1.77), 12

Internal analysis (probable high exposure)

measures of exposure duration Few long-term workers Short duration of follow-up The workers at companies with ≥ 50% styrene-exposed workers were included in the European study by Kogevinas et al. (Kogevinas et al. 1994a, Kogevinas et al. 1993)


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments (Kolstad et al. 1995) IRR (95% CI), no. of observed cases Pancreatic cancer overall 2.2 (1.1–4.5), 17

Years of exposure < 1 3.1 (0.9–10.3), 9 ≥ 1 3.4 (1.0–11.4), 8

Year of first employment 1970 or before 2.0 (0.6–6.9), 10 after 1970 1.4 (0.4–4.7), 7

Other cancers with elevated SIRs No consistent findings in internal analyses by first year of employment, employment duration, and latency

Kolstad et al. 1996

Denmark

nested case-control study of myeloid leukemia

Cohort: Kolstad et al. 1994, 1995 Cases: 12 myeloid leukemia patients with clonal chromosome aberrations (chromosome analysis was available on 19 of the myeloid leukemia cases in the cohort) Controls: 57 randomly selected employees without styrene exposure and matched to cases by age (3 per

See Kolstad et al. 1994, 1995

OR (95% CI) for myeloid leukemia with clonal chromosome aberrations Any exposure 2.5 (0.2–25.0)

Probability of exposure low 3.0 (0.3–32.2) high 1.6 (0.1–22.0)

Years of exposure < 1 5.9 (0.5–74.3) ≥ 1 1.1 (0.1–15.3)

Years since first exposure < 10 no exposed cases ≥ 10 3.7 (0.4–40.3)

Year of first employment

Small numbers of exposed cases


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments case) ORs calculated by conditional and unconditional regression that included probability of styrene exposure (none, low, high), year of first employment, time since first employment, age, and year of diagnosis

1970 or before 5.9 (0.6–57.8) after 1970 2.3 (0.2–26.2)

Kogevinas et al. 1994a, Kogevinas et al. 1993 Europe

historical cohort follow-up varied by country 1945–91 avg. 13 yr 539,479 person-years

40,688 workers (85% men) at 660 reinforced-plastics plants in Denmark (39%), Finland (5%), Norway (5%), Italy (18%), Sweden (9%), and U.K. (24%) 60% worked < 2 yr, 9% worked ≥ 10 yr SMRs based on national rates Poisson regression models used to compare exposure-specific cancer rates (RR) and conduct trend analyses for exposure within the

Job titles were used to assign workers to the following exposure categories: laminators (26%) unspecified tasks (48%) other exposed jobs (13%) non-styrene exposed (10%)unknown job titles (3%) Exposure matrix was constructed from 16,500 personal styrene measurements obtained 1955–90 and 18,500 measurements of urinary metabolites in the 1980s Cumulative styrene exposure and average exposure estimated for each subject from job

Total cohort Cancers with non-significant excess mortaltiy (> 10%) small intestine 1.50 (0.31–4.38), 3 larynx 1.11 (0.53–2.05), 10 ovary 1.40 (0.70 – 2.51), 11 myeloid leukemia 1.10 (0.63 – 1.79), 16 Cancers with significantly decreased mortality all cancer 0.87 (0.81–0.94), 686 buccal cavity and pharynx 0.33 (0.11–0.77), 5 rectum 0.62 (0.38–0.95), 21 breast 0.52 (0.28–0.89), 13 brain 0.62 (0.37–0.98), 18

Laminators Cancers with non-significant excess mortality small intestine 2.27 (0.06–12.66), 1 pancreas 1.48 (0.76–2.58), 12 larynx 1.55 (0.32–4.52), 3

Imprecise measures of cumulative styrene exposure and short duration of follow-up limit power to detect an effect Study population included the Danish workers (39%) (Kolstad et al. 1994, 1995)


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Study



Exposure Assessment

Levels Effects

SMR (95% CI), no. of observed deathsb Comments population records and exposure

levels averaged across country, period, and job Exposure levels among laminators declined from ~200 ppm before 1965 to < 80 ppm in the 1980s

ovary 2.61 (0.71–6.69), 4 thyroid 2.27 (0.06–12.66), 1 NHL 1.40 (0.56–2.88), 7 Hodgkin’s disease 1.33 (0.27–3.88), 3

Poisson regression exposure models for cancer Test for trend in RRs: P-value Average exposure LH 0.019 leukemia 0.47 malignant lymphoma 0.052

Time since first exposure LH 0.012 leukemia 0.094 malignant lymphoma 0.072

Cumulative exposure no increase in RR with increasing exposure for any LH cancer type Pancreas RR increased with increasing cumulative exposure (P = 0.068) Esophagus, or kidney RR increased (non-significantly) with cumulative exposure (slightly for esophagus) Lung No increase in RR with increasing cumulative exposure

CI = confidence interval, IRR = incidence rate ratio, LH = lymphohematopoietic cancer, OR = odds ratio, RR = rate ratio, SIR= standard incidence ratio, SMR = standard mortality ratio. aThe table contains results from the latest update of a study population. Separate entries are made for related studies if there were major differences between publications, such as differences in the study design (e.g., nested case control and cohort) or population composition. The results of the earlier publication for the


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8-company U.S. cohort (Wong et al. 1990) for respiratory cancer are also presented in addition to the later publication (Wong et al. 1994) since the excess of respiratory cancer was the basis for the nested case-control study. bUnless otherwise stated. cNot including rectum. dRecords and follow-up from one company were incomplete, so the analysis for that company was reported separately.


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3.2 The styrene-butadiene rubber industry 1

Generally lower styrene exposure levels are seen in the styrene-butadiene rubber industry

than the glass fiber–reinforced plastics industry, although significant exposures to

workers can still occur (see Section 2.6). Mean levels reported for this industry are

generally less than 15 ppm for synthesis of styrene-butadiene latex, and concentrations

below 0.15 ppm have been reported for vulcanization and extrusion processes involving

styrene-butadiene rubber. Exposure to end-users (such as rubber tire manufacturers)

would likely be even lower. Workers in the styrene-butadiene industry can be exposed to

1,3-butadiene and DMDTC, in addition to styrene (Delzell et al. 2001, Macaluso et al.

2004). This section does not include studies on end-users except for McMichael et al.

(1976a) because it provides specific estimates for workers in a plant producing styrene

butadiene (primarily) and other rubbers.

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4

5

6

7

8

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12

13

14

15

16

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27

28

McMichael et al. (1976a) studied cancer mortality at a rubber tire manufacturing plant in

the United States. Meinhardt et al. (1982, 1978) and Lemen et al. (1990) initially studied

2,756 workers at two styrene-butadiene rubber plants (forming one complex) in Texas.

Matanoski and coworkers (Matanoski et al. 1997, Matanoski et al. 1993, Matanoski et al.

1990, Matanoski and Schwartz 1987, Santos-Burgoa et al. 1992) studied workers (from

12,110 to 13,686, depending on study)2 in eight other styrene-butadiene rubber plants

(seven U.S. plants and one Canadian plant). Later, Delzell and colleagues (Delzell et al.

1996, 2001, 2006, Macaluso et al. 2004, 1996; Sathiakumar et al.1998, 2005, and Graff

et al. 2005) studied 13,130 to 16,610 styrene-butadiene rubber industry workers from the

same plants studied by Meinhardt et al. and seven of the plants studied by Matanoski,

Santos-Burgoa, and coworkers (Delzell et al. 2001, Delzell et al. 1996). Delzell et al.,

Macaluso et al. (1996, 2004), Sathiakumar et al. (1998, 2005), and Graff et al. (2005) did

not have access to information from the previous studies by Meinhardt et al., Matanoski

et al., and Santos-Burgoa et al. that allowed identification of individual subjects and a

formal evaluation of the overlap between the populations. The study populations were

established by different procedures and exclusion criteria, which may partly explain the

2 Number of workers varied among the publications.


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lack of complete consistency in the number of study subjects across the populations. An

overview of the studies is presented in Table 3-4

1

2

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

23

24

25

26

27

28

29

30

3.2.1 United States: McMichael et al. 3

McMichael et al. (1976a) studied 6,678 male workers at a rubber tire manufacturing

plant. A small fraction of the total cancer cases (2% with 5 or more years of employment

and 3% with 2 or more years of employment) was observed among workers engaged in

the production of styrene-butadiene and other synthetic rubbers; however, the proportion

of the total cohort exposed to synthetic rubbers was not given. The total population was

followed from 1964 to 1972, deaths were identified, and work histories were compared

between cases and an age-stratified random sample of all workers (22%) in internal

analyses. Work histories were extracted from personnel records and grouped into 16

major work areas. One of these areas was work in the synthetic plant where styrene-

butadiene rubber was produced. For workers with at least 5 years employment in the

synthetic plant, significantly increased risk ratios were observed for stomach cancer (RR

= 2.2 , 99.9% CI = 1.4 to 4.3, number of deaths not stated), lymphohematopoietic cancer

(RR = 6.2, 99.9% CI = 4.1 to 12.5), and lymphatic leukemia (RR = 3.9, 99.9% CI = 2.6

to 8.0). No other significant associations between work in synthetic rubber production

and other selected cancers were observed. [Note: exposure ratios and/or risk ratios were

reported by the authors only for certain selected cancer sites (see Table 3-9). It appears

that if an exposure ratio greater than 1 was observed, an age-standardized risk ratio (in

comparison with all workers in the cohort) was then calculated and reported.]

3.2.2 United States: Meinhardt et al. 22

Meinhardt et al. studied the mortality of 2,756 styrene-butadiene rubber workers at two

plants in Texas, prompted by the deaths of 2 workers from leukemia (Meinhardt et al.

1982, 1978). White male workers with at least six months of non-management or non-

administrative employment were included in the study. The population was followed

from 1943 through 1976, and 3% were lost to follow-up. SMR analyses compared

observed deaths with expected values computed from the national rates. The average

length of employment was about 10 years. A total of 53,929 person-years were

accumulated, and the average follow-up was 19 years. Average TWA styrene exposure


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levels based on samples from all areas of the production facilities were 0.94 ppm (0.03 to

6.46) and 1.99 ppm (0.05 to 12.3) for the two plants.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

27

28

29

Among all workers, 56 cancer deaths were recorded [SMR = 0.72, 95% CI = 0.54 to

0.93]. Statistically nonsignificant increases in mortality were seen for all

lymphohematopoietic malignancies [SMR = 1.32, 95% CI = 0.66 to 2.37, 11 deaths],

NHL 1.65 [95% CI = 0.45 to 4.27, 4 observed deaths], and leukemia 1.73 [95% CI = 0.63

to 3.73, 6 observed deaths]. [The bracketed SMRs for both plants combined were

calculated using the data provided in the publication for each plant.] Because most

leukemia mortality in one of the plants was among workers who had started work before

the end of 1945, a separate analysis was conducted for all 600 workers who started work

before the end of 1945; this analysis showed a statistically significant increase in

mortality for all lymphohematopoietic malignancies (SMR = 2.12, P < 0.05, 9 observed

deaths). No subanalyses were conducted by level of estimated styrene exposure.

Lemen et al. (1990) reported on a further follow-up of this cohort through December 31,

1982 at one plant (A) and December 31, 1981 for a second plant (B), yielding a total of

43,341 person-years at risk of death and 390 observed deaths, 77 from cancers, at plant A

and 26,314 person-years and 148 observed deaths, 29 from cancers, at plant B. In the

subcohort exposed to the batch production process used between 1943 and 1945, a total

of 19,582 person-years and 291 observed deaths, 61 from cancers, were available for

analysis. No SMRs for cancers were reported in this follow-up report. However, the

authors noted that mortality for cancers of the trachea, bronchus, and lung had increased

(from 16 to 34 deaths) and that the only other increases in SMRs were observed for

lymphosarcoma and reticuloma (3 deaths in the first analysis and 5 deaths in the follow-

up; these 2 additional deaths occurred in the subcohort exposed to the batch process, as

did the one additional lymphohematopoietic death in this follow-up.

3.2.3 United States and Canada: Matanoski, Santos-Burgoa, and coworkers 26

Matanoski and coworkers established a cohort of male workers employed for more than 1

year in seven U.S. styrene-butadiene rubber plants and for more than 10 years in one

Canadian styrene-butadiene rubber plant between 1943 and 1976 (Matanoski et al. 1997,


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Matanoski et al. 1993, Matanoski et al. 1990, Matanoski and Schwartz 1987, Santos-

Burgoa et al. 1992). The number of workers included in the different publications

differed slightly, from 12,110 (Matanoski et al. 1997, Matanoski et al. 1993, Matanoski

et al. 1990), to 13,686 (Santos-Burgoa et al. 1992), or 13,920 (Matanoski and Schwartz

1987). The population was initially followed to 1979 (Matanoski and Schwartz 1987) and

was updated through 1982 (Matanoski et al. 1990). Loss to follow-up was 3%, and

251,431 person-years were accumulated; the average follow-up was about 21 years.

Mortality was compared with national rates, and SMR values were calculated (Matanoski

et al. 1990, Matanoski and Schwartz 1987). In internal analyses of lymphohematopoietic

malignancies, odds ratios were estimated by Mantel-Haenszel methods and by

conditional and unconditional regression analysis (Matanoski et al. 1997, Santos-Burgoa

et al. 1992). Initial internal analyses relied on 59 cases and 193 controls individually

matched by plant (and other variables) (Santos-Burgoa et al. 1992). Subsequent analyses

included 58 cases and replaced the original controls with 1,242 controls sampled without

individual matching (Matanoski et al. 1997). This was done to avoid over-matching,

because measurements indicated that styrene exposure levels differed between plants.

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A panel of experts constructed a job-exposure matrix for internal analyses (Santos-

Burgoa et al. 1992). From the job titles listed in the personnel records, cases and controls

were classified as exposed or not exposed to styrene and butadiene and were assigned a

relative exposure rank (0 to 10, 10 representing the highest exposure). Cumulative

exposure was calculated from duration of employment in each job, and cases and controls

were classified as having a cumulative exposure score above or below the geometric

mean value.

Later, five of the eight plants provided 3,649 measurements of styrene in work-room air

taken between 1978 and 1983 (Matanoski et al. 1993). The average styrene level for all

plants was 3.53 ppm (SD = 14.32) and varied between 0.29 ppm and 6.66 ppm across the

plants. Styrene levels were averaged across jobs and plants, and average cumulative

styrene exposure levels were estimated for cases and controls from information about

plant, job title, and number of months exposed (Matanoski et al. 1997).


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Among all workers at the eight plants, overall cancer mortality was less than expected

(SMR = 0.85, 95% CI = 0.78 to 0.93, 518 observed deaths). Statistically nonsignificant

increases in mortality were observed for Hodgkin’s disease (SMR = 1.20, 95% CI = 0.52

to 2.37, 8 observed deaths) and for other lymphatic malignancies (SMR = 1.11, 95% CI =

0.64 to 1.77, 15 observed deaths). No increased mortality was reported for the other

lymphohematopoietic cancers, including leukemia (Matanoski et al. 1990). Mortality

from all lymphopoietic [lymphohematopoietic] malignancies was not related to duration

of employment. SMR values were also presented separately for white and black

production workers, maintenance workers, utility workers, and other workers, but it was

not clear whether styrene exposure differed among these categories.

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Among workers categorized as having a cumulative styrene exposure score above

average, the internal matched analyses presented by Santos-Burgoa et al. (1992) showed

statistically nonsignificant increases in the odds ratios for leukemia (OR = 3.13, 95% CI

= 0.84 to 11.2, 26 cases, 84 controls), lymphosarcoma (OR = 1.33, 95% CI = 0.11 to

16.6, 6 cases, 23 controls), and other lymphatic malignancies (mainly myeloma) (OR =

1.35, 95% CI = 0.25 to 7.40, 18 cases, 56 controls), but a non-significant decrease in the

OR for Hodgkin’s disease (OR = 0.40, 95% CI = 0.05 to 3.25, 8 cases, 29 controls).

Comparable ORs were seen in unmatched analyses of the same dataset. In matched

models that controlled for exposure to butadiene, no increased risk was apparent for

leukemia (OR = 1.06, 95% CI = 0.23 to 4.95), or other lymphatic malignancies (OR =

0.94, 95% CI = 0.16 to 5.53); the OR for all lymphohematopoietic malignancies was 1.29

(95% CI = 0.53 to 3.15). There was no indication of positive interaction between

exposure to styrene and butadiene for all lymphohematopoietic malignancies; however,

no results were obtained for leukemia, because the model did not converge.

Matanoski et al. (1997) and colleagues presented updated analyses that relied on non-

matched controls and measurement-derived estimates of styrene exposure. Analyses were

based on average or cumulative styrene exposure levels (calculated across all exposed

years). Using a step-down unconditional logistic regression with age, age at first hire,

race, year of hire before 1950, and both styrene and butadiene in the initial model, a time-

weighted working lifetime average styrene exposure level of 1 ppm increased the ORs for


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myeloma (OR = 3.04, 95% CI = 1.33 to 6.96, 10 deaths), lymphomas (OR = 2.67, 95%

CI = 1.22 to 5.84, 12 deaths), and all lymphohematopoietic malignancies (OR = 2.20,

95% CI = 1.46 to 3.33, 58 deaths), but not for leukemia (no estimate was provided for

leukemia). [Note that styrene alone remained in the final model for each of these

cancers.] Also, final models for leukemia and Hodgkin’s disease (which were not

associated with styrene exposure) included only exposure to butadiene. With respect to

cumulative exposure, using the same initial variables, the mortality for leukemia

increased statistically significantly by increasing cumulative styrene exposure (P = 0.006)

in a final model in which both butadiene exposure and duration of employment remained.

Styrene alone was also significantly associated with myeloma (P = 0.023) and with all

lymphohematopoietic cancers (P = 0.000 [P-value as reported in the paper]) in a final

model in which styrene and duration of employment remained. [Final models for

Hodgkin’s disease included butadiene exposure and duration. Note that the ICD codes for

lymphomas (200 & 202) are the same as non-Hodgkin’s lymphoma.]

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3.2.4 United States and Canada: Delzell, Sathiakumar, Macaluso, Graff 15

Delzell et al. (1996) established a cohort of all 17,964 male workers employed for at least

one year between 1943 and 1990 at eight U.S. styrene-butadiene rubber plants (two of

these plants were organized as one company complex) and one Canadian styrene-

butadiene rubber plant. (The start date for some of the plants varied between 1950 and

1965.) The population included workers at seven of the eight plants previously studied by

Matanoski et al. (1993, 1990) and Santos-Burgoa et al. (1992) and at the two-plant

complex studied by Meinhardt et al. (1982). The companies employed an estimated total

of 25,500 workers. [Note that the Delzell cohort exanded the cohort to include more

recent employees with start dates up to 1990, whereas the earlier cohorts followed

workers employed from 1943 to 1976. Adding workers with lower exposures, shorter

latency, and duration worked might reduce apparent risk.]

One series of studies reporting findings (SMRs) for multiple cancers sites (Sathiakumar

et al. 1998, Sathiakumar et al. 2005) or specifically for lymphopoietic cancers (Delzell et

al. 1996) for the entire cohort, and for subcohorts based on work areas

(lymphohematopoietic cancer only) whereas a second series of studies reported findings


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(RR) based on quantitative estimates of exposure to styrene (and butadiene or DMDTC)

and mortality from lymphohematopoietic cancers (Delzell et al. 2001, Graff et al. 2005,

Macaluso et al. 1996). A full report of the latest update (Graff et al. 2005, Satiakumar et

al. 2005) of the cohort was published by Delzell et al. in 2006. This report contains

details of some analyses that were not included in the individual papers.

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3.2.4.1 Study population 6 The first study was conducted among workers employed for at least one year between

1943 and 1990 and followed up until 1991 (Delzell et al. 1996, Sathiakumar et al. 1998);

at that time, 25% had died (3,976), 70% were presumed alive, and 5% were lost to

follow-up. Mortality analysis was conducted on 15,649 subjects and excluded 2,315

Canadian workers who had not worked in styrene-butadiene rubber or other related

operations or who had worked in unspecified areas of one of the plants. Delzell et al.

2006 reported that the “2,312” Candadian workers had worked in butyl rubber production

or styrene production or were unspecified. Follow-up was later extended to 1998 for the

cohort of 17,924 workers, and included 6,327 observed deaths while vital status was

unknown for 570 (3%) (Delzell et al. 2006, Sathiakumar et al. 2005). The authors stated

that 40 workers from the orignal cohort were excluded because they did not meet study

criteria (employment length or gender), or were duplicates, [but they did not discuss the

2,315 non-styrene-butadiene rubber workers excluded from the first study]. Analyses

based on work areas were limited to workers in styrene-butadiene rubber operations (N =

15,612).

Different subsets of the cohort were used in the series of studies of quantitative estimates

of styrene exposure and lymphohematopoietic cancer mortality (primarily leukemia)

(Delzell et al. 2001, Delzell et al. 2006, Graff et al. 2005, Macaluso et al. 1996). The

analyses reported by Macaluso et al. (1996) and Delzell et al. (2001) were based on the

1991 follow-up, and analyses reported by Graff et al. and Delzell et al. 2006 were based

on the 1998 follow-up. The study by Macaluso et al. (1996) (which evaluated cumulative

exposure to styrene, butadiene and mortality from leukemia) excluded 1,354 workers (of

the 17,964 member cohort) at two plants for whom quantitative estimates of exposure

could not be established, and the analyses were based on 16,610 workers. Delzell et al.


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(2001) further excluded twelve workers with duplicate records and 3,468 workers who

died before reaching 40 years of age or 10 years latency, because no leukemia deaths

occurred in these groups; this left 13,130 workers. This study also used a revised

exposure assessment (see below) and evaluated leukemia mortality and quantitative

exposure to styrene, butadiene, and DMDTC. The study reported by Graff et al. (2005)

and Delzell et al. (2006) stated that their analysis was on 16,579 workers for whom

quantitative estimates of exposure could be established (which excluded 25 workers

(from Macaluso et al. 2006) who were determined to have duplicate records or did not

meet study criteria such as employment length or gender).

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Vital status was established for U.S. workers via plant records, the National Death Index

and DMV records, and in Canada, through plant personnel and benefit records and record

linkage with the Canadian Mortality Database. [Note that Matanoski et al. (1990)

reported that they relied on company pension and insurance records to identify deaths

among employees who worked 10 years or more or reached age 45 during employment

because of the high cost of a death search through Statistics Canada for the workers.]

3.2.4.2 Exposure assessment and job classification 16 Personnel records were reviewed, and 308 work-area groups or job groups with similar

tasks and exposure potential were identified. The groups were further combined into five

main process groups and seven process subgroups: (1) rubber/butadiene production

(37%): polymerization, coagulation, finishing; (2) maintenance (24%): shop, field;

(3) labor (15%): production, maintenance; (4) laboratories (9%); and (5) other operations

(15%) (Sathiakumar et al. 1998). Macaluso et al. (1996) constructed a plant-specific, job-

exposure matrix from industrial hygiene monitoring surveys, archival material, walk-

through surveys, meetings with plant officials, and interviews with workers. Eight-hour

TWA exposure levels for styrene, butadiene, and benzene were estimated for each year

between 1943 and 1992 for each of the work areas or job groups by air dispersion models

(Macaluso et al. 1996). Cumulative exposure was computed, taking into account the

extents and durations of different tasks. As of the end of the 1991 follow-up, 83% of the

cohort were considered to have been exposed to styrene, with a median cumulative


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exposure of 7.4 ppm-years, and 75% to butadiene, with a cumulative exposure of 11.2

ppm-years (Macaluso et al. 1996).

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Delzell et al. (2001) later characterized this exposure estimation process as controversial,

and they revised the exposure estimates and added estimates of exposure to DMDTC, as

further described by Macaluso et al. (2004). The authors did not substantiate this critique,

but others have cited them as characterizing the original estimates as uncertain and not

completely validated (Sielken and Valdez-Flores 2001). [The revised exposure estimates

used by Macaluso et al. (2004) represented an improvement over the original estimates

because of detailed industrial hygiene and chemical engineering reviews of the processes,

job activities and work area, and historical changes. A senior industrial hygienist, who

had extensive experience within the industry and with the methodology for estimating

historical exposures, guided the work, which included identification of new tasks,

additional information on the operations, modification of some of the assumptions needed

to estimate exposure, and verification that all of the assumptions were reasonable.

Information on use of personal protective equipment was obtained through interviews

with long-term employees.] According to the original exposure assessment, estimated

TWA styrene exposure levels for active workers declined from 1.8 ppm in the 1940s to

0.1 ppm in the early 1990s (Macaluso et al. 2004), partly because of decreasing exposure

levels and partly because of decreasing styrene exposure prevalence. The revised styrene

TWA exposure estimates were about twice as high as the original estimates reported in

Macaluso et al. 1996 and declined from about 2 ppm during 1940 to 1970 to about

0.5 ppm in the late 1980s. The revised assessment estimated a median cumulative

exposure of 17 to 18 ppm-years (Delzell et al. 2001, Macaluso et al. 2004) for the 85% of

the workers who were exposed to styrene. The cumulative styrene exposure estimates

were highly correlated with those for butadiene and DMDTC (Spearman rank

correlations of 0.78 and 0.60, respectively). Note that 79% of workers were estimated to

have been exposed to butadiene and 62% to DMDTC (Delzell et al. 2001). Exposure to

DMDTC occurs primarily though dermal absorption, and cumulative estimated exposure

was calculated as mg-years DMDTC/cm of skin.


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3.2.4.3 SMR analyses 1 SMR analyses compared observed with expected deaths calculated from national

mortality rates (Delzell et al. 2006, Delzell et al. 1996, Sathiakumar et al. 1998,

Sathiakumar et al. 2005).

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SMR analyses based on follow-up until 1991 showed that among all styrene-butadiene

rubber workers, overall cancer mortality was less than expected (SMR = 0.93, 95% CI =

0.87 to 0.99, 950 observed deaths) (Sathiakumar et al. 1998). Significant deficits in

mortality were also seen for cancer of the buccal cavity and pharynx (SMR = 0.50, 95%

CI = 0.28 to 0.82, 15 observed deaths) and esophagus (SMR = 0.59, 95% CI = 0.35 to

0.93, 18 observed deaths). A statistically nonsignificant excess of mortality from

leukemia was seen among all workers (SMR = 1.31, 95% CI = 0.97 to 1.74, 48 observed

deaths), and significant excesses were seen among all workers ever employed hourly

(SMR = 1.43, CI not reported, P < 0.05, 45 deaths) and among ever-hourly workers with

employment duration of at least 10 years and latency of at least 20 years (SMR = 2.24,

95% CI = 1.49 to 3.23, P < 0.05, 28 deaths) (Sathiakumar et al. 1998). Increased

leukemia mortality was also seen among workers in polymerization (SMR = 2.51, 95%

CI = 1.40 to 4.14, 15 deaths), coagulation (SMR = 2.48, 95% CI = 1.00 to 5.11, 7

observed deaths), the maintenance subgroup of labor (SMR = 2.65, 95% CI = 1.41 to

4.53, 13 observed deaths), and laboratories (SMR = 4.31, 95% CI = 2.07 to 7.93, 10

observed deaths) (Delzell et al. 1996).

Repeated SMR analyses based on the extended follow-up until 1998 did not change this

mortality pattern considerably (Sathiakumar et al. 2005). All cancer mortality (SMR =

0.92, 95% CI = 0.88 to 0.97, 1,608 observed deaths), and buccal cavity-pharynx cancer

mortality (SMR = 0.47, 95% CI = 0.29 to 0.71, 22 observed deaths), still showed

significant deficits, while this no longer was the case for esophageal cancer (SMR = 0.94,

95% CI = 0.68 to 1.26, 44 observed deaths). With respect to lymphohematopoietic

cancers, non-significant excesses in mortality were observed for leukemia (SMR = 1.16,

95% CI = 0.91 to 1.47, 71 observed deaths) and Hodgkin’s disease. All

lymphohematopoietic malignancies, NHL, and multiple myeloma showed observed

numbers of death close to the expected (Sathiakumar et al. 2005). Sub-analyses indicated


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statistically nonsignificant increases in leukemia in workers ever employed hourly (SMR

= 1.23, 95% CI = 0.94 to 1.57, 63 observed deaths), and significant excesses among those

employed for at least 10 years and 20 to 29 years since hire (SMR = 2.58, 95% CI = 1.56

to 4.03, 19 observed deaths). No excess of leukemia was seen for 30 years or more after

first employment (SMR = 1.02, 95% CI = 0.62 to 1.58, 20 observed deaths).

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Several analyses of cell-type specific leukemias were conducted by Sathiakumar et al.

(2005), Graff et al. (2005), and Delzell et al. (2006) including acute lymphocytic

leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia

(AML), chronic myelogenous leukemia (CML) and other leukemias and NHL+CLL. A

statistically significant increase in SMR for CML was observed among the ever-hourly

employed workers (SMR = 2.00, 95% CI = 1.00 to 3.58, 11 observed deaths)

(Sathiakumar et al. 2005). A statistically nonsignificant increase in mortality from CLL

was observed (SMR = 1.71, 95% CI = 0.96 to 2.81, 15 observed deaths), while there was

no increase in the numbers of AML (SMR = 0.97, 95% CI = 0.48 to 1.73, 11 observed

deaths) or ALL (SMR = 0.51, 95% CI = 0.01 to 2.82, 1 observed death). Statistically

significant increases were observed for AML among workers with < 20 years since hire

and < 10 years employment (SMR = 4.78, 95% CI = 1.30 to 12.24, 4 deaths) and for

CML (SMR = 6.55, 95% CI = 2.4 to 14.26, 6 deaths). Significant (or borderline)

increases were also found for NHL+CLL among every-hourly workers (SMR = 1.30,

95% CI + 0.99 to 1.67, 60 observed deaths), workers with 20 to 29 years since hire and

10+ years employment (SMR = 1.90, 95% CI = 1.01 to 3.25, 13 observed deaths), and

with 30+ years since hire and 10+ years employment (SMR = 1.49, 95% CI = 1.02 to

2.10, 32 observed deaths).

With respect to work area and job type, statistically significant excesses of all leukemias

were observed for workers employed in polymerization (SMR = 2.04, 95% CI = 1.21 to

3.22, 18 observed deaths), coagulation (SMR = 2.31, 95% CI = 1.11 to 4.25, 10 observed

deaths), maintenance labor (SMR = 2.03, 95% CI = 1.14 to 3.35, 15 observed deaths) and

laboratories (SMR = 3.26, 95% CI = 1.78 to 5.46, 14 observed deaths) (which appear to

be due primarily to increases in CLL in the same departments; the SMRs for CLL were

4.97 (95% CI = 2.15 to 9.80, 8 observed deaths) in polymerization; 6.07 (95% CI = 1.97


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to 14.17, 5 observed deaths), for coagulation; 3.09 (95% CI = 0.84 to 7.92, 4 observed

deaths) in maintenance labor; and 5.59 (95% CI = 1.52 to 14.31, 4 observed deaths) in

laboratories. SMR also were increased for AML (SMR = 2.95, 95% CI = 0.96 to 6.88, 5

observed deaths) in maintenance labor, CML in laboratories (SMR = 5.22, 95% CI = 1.08

to 15.26, 3 observed deaths), and CLL among finishers (SMR = 3.44, 95% CI = 1.38 to

7.09, 7 observed deaths). (The authors noted that while workers were assigned to one

department for these analyses, there was a considerable likelihood of overlap between

various departments (Sathiakumar et al. 2005, Delzell et al. 2006). Significant increases

were also observed for NHL+CLL among workers in polymerization (SMR = 2.18, 95%

CI = 1.31 to 3.41, 19 observed deaths) and finishing (SMR = 1.91, 95% CI = 1.21 to

2.86, 23 observed deaths).

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Graff et al. (2005) and Delzell et al. (2006) also reported SMRs, adjusted for age, race,

and calendar year, for all leukemias, CLL, AML, CML, and other leukemias, NHL, and

multiple myeloma (MM), by cumulative level of styrene exposure. Statistically

significant increases in SMRs were observed in the two highest categories of styrene

exposure for all leukemias: SMR = 1.87 (95% CI = 1.02 to 3.13, 14 observed deaths) for

31.8 to < 61.1 ppm-years, and SMR = 1.91 (95% CI = 1.09 to 3.10, 16 observed deaths)

for 61.1+ ppm-years. Statistically significant increases also were seen for the highest

category of exposure only for NHL (SMR = 1.97, 95% CI = 1.08 to 3.31, 14 observed

deaths), CLL (SMR = 3.10, 95% CI = 1.01 to 7.24, 5 observed deaths), and for the

highest category for NHL+CLL (SMR = 2.29, 95% CI =1.36 to 3.62, 18 observed

deaths).

3.2.4.4 Internal analyses of leukemia and other lymphohematopoietic cancers 23 Among the 15,649 workers studied by Delzell et al. (1996), 48 deaths with leukemia as

the underlying diagnosis had been identified as of the end of follow-up in 1991. [This

analysis excluded the 2,315 non-styrene-butadiene rubber workers.] Macaluso et al.

(1996) included 58 leukemia deaths (7 with leukemia as a contributory diagnosis and 51

with leukemia as an underlying diagnosis on the death certificate) identified among

16,610 workers followed up to 1991. In a later analysis, Delzell et al. (2001) added 1

decedent with myelodysplasia as the underlying cause of death and a medical record that


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indicated leukemia. In the follow-up of 17,924 men to 1998, Sathiakumar et al. (2005)

identified a total of 162 lymphohematopoietic cancers: 53 NHL, 12 Hodgkin’s disease,

71 leukemia and 26 multiple myeloma based on the underlying cause of death. In the

analysis of 16,579 workers followed to 1998 for whom quantitative exposure estimates

were available, Graff et al. (2005) identified 81 deaths from leukemia, 58 from NHL, 27

from multiple myeloma, and 13 from Hodgkin’s disease, with these diagnoses as the

underlying or contributing cause of death and confirmed by medical records, if available.

(Note: Delzell et al. 2006 stated that the death certificate diagnoses and ICD codes (e.g.,

71 leukemias) were used for the external analysis to avoid information bias).Relative

risks were computed by Poisson regression models (Delzell et al. 2001, Delzell et al.

2006, Graff et al. 2005, Macaluso et al. 1996) or the Mantel-Haenszel method (Macaluso

et al. 1996).

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Internal analyses of lymphohematopoietic cancers used several approaches, resulting in a

large number of statistical analyses. Relative risks were calculated for quartiles of

cumulative (total ppm-years) of styrene exposure and ppm-years of exposure due to

peaks above 50 ppm or below 50 ppm using single-chemical (styrene), two-chemical

(styrene + butadiene or styrene + DMDTC), or three-chemical ( styrene + butadiene +

DMDTC) models. Models were adjusted for age and time since hire. In addition, analyses

were also conducted using cross-categories of different levels of cumulative styrene or

butadiene exposure.

Macaluso et al. (1996) presented rate ratios (relative risks) for leukemia mortality by

cumulative exposure to styrene based on the original exposure assessment and adjusted

for exposure to butadiene. Although SMRs for leukemia in the cohort tended to increase

with increasing cumulative styrene exposure, the internal analyses that controlled for

butadiene exposure showed no significant trend (Macaluso et al. 1996). The findings of

the internal analysis were as follows: 0 ppm-year (reference group), RR = 1; 1 to 4 ppm-

years, RR = 0.9; 5 to 9 ppm-years, RR = 5.4; 10 to 39 ppm-years, RR = 3.4; ≥ 40 ppm-

years, RR = 2.7 (P for linear trend = 0.14). The authors also evaluated the association

between benzene and leukemia and reported a weak association between increasing

levels of cumulative exposure to benzene and leukemia mortality rates; that association


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was eliminated when they controlled for exposure to butadiene and styrene. The authors

concluded that no association existed between benzene and leukemia and excluded

benzene from their other analyses.

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Delzell et al. (2001) presented RRs for leukemia mortality by cumulative exposure to

styrene based on the revised exposure assessment. As mentioned previously, the analysis

excluded workers who died before reaching 40 years of age or 10 years latency because

no deaths from leukemia occurred before this age and length of employment. Controlling

for age and time since hire, Delzell et al. also found an increasing trend of leukemia

deaths with cumulative styrene exposure. The findings were as follows: 0 ppm-years

(reference group), RR = 1.0; > 0 to < 20.6 ppm-years, RR = 1.2 (95% CI = 0.5 to 3.3);

20.6 to < 60.4 ppm-years, RR = 2.3 (95% CI = 0.9 to 6.2); ≥ 60.4 ppm-years, RR = 3.2

(95% CI = 1.2 to 8.8). This trend was reduced when butadiene exposure was introduced

to the model; the findings were as follows: 0 ppm-years (reference group), RR = 1.0; > 0

to < 20.6 ppm-years, RR = 1.1 (95% CI = 0.3 to 4.0); 20.6 to < 60.4 ppm-years, RR = 1.6

(95% CI = 0.4 to 6.4); ≥ 60.4 ppm-years, RR = 1.8 (95% CI = 0.4 to 7.3). If analyses

furthermore included DMDTC exposure, no increasing trend was seen; the findings were

as follows: 0 ppm-years, RR = 1.0; > 0 to < 20.6 ppm-years, RR = 0.6 (95% CI = 0.1 to

2.5); 20.6 to < 60.4 ppm-years, RR = 0.8 (95% CI = 0.2 to 3.7); ≥ 60.4 ppm-years, RR =

0.8 (95% CI = 0.2 to 3.8).

Among workers with a cumulative butadiene exposure below 20 ppm-years (the worker

category with the lowest butadiene exposure according to the original exposure

assessment), the risk of leukemia increased with increasing cumulative exposure to

styrene. The findings were as follows: 0.1 to 9 ppm-years (reference), RR = 1.0; 10 to 39

ppm-years, RR = 1.7 (95% CI = 0.5 to 6.0); ≥ 40 ppm-years, RR = 7.0 (95% CI = 2.2 to

22) (Macaluso et al. 1996). No such trend was seen for strata with higher levels of

cumulative butadiene exposure. Two deaths from leukemia occurred among styrene-

exposed workers with no exposure to butadiene, but no formal risk assessment was

conducted for this category of workers. No deaths from leukemia occurred among

workers exposed to butadiene but not to styrene. No trend with cumulative styrene

exposure was seen among the workers with the lowest cumulative butadiene exposure


122 9/29/08

(< 38.7 ppm-years) according to the revised exposure estimates, but this category

included only 4 workers with styrene exposure above the reference category (Delzell et

al. 2001). On the other hand, among workers classified with the highest cumulative

butadiene exposure (≥ 287.3 ppm-years), the risk of leukemia increased with cumulative

styrene exposure; the findings were as follows: 10.4 to 40.5 ppm-years, RR = 2.6 (95%

CI = 0.7 to 9.2, 3 observed deaths); ≥ 40.6 ppm-years, RR = 4.1 (95% CI = 2.0 to 8.4, 18

observed deaths). No deaths from leukemia occurred in the reference category (< 10.4

ppm-years).

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

Graff et al. (2005) repeated these analyses based on the revised exposure assessment by

(Delzell et al. 2001) and the 81 leukemia deaths observed up to the end of follow-up in

1998 (Graff et al. 2005). In addition, Graff et al. analyzed these leukemias by subtype:

CLL (25 deaths); AML (including myelogenous and monocytic leukemias) (26 deaths);

CML (16 deaths), and other leukemias (14 deaths). Graff et al. also analyzed findings for

other lymphohematopoietic cancers, including NHL (58 deaths), multiple myeloma (27

deaths), and Hodgkin’s disease (13 deaths). Detailed descriptions of the methods and full

models included in these analyses were reported in Delzell et al. 2006. Findings for the

single-chemical, two-chemical and three-chemical models and leukemia, NHL and

NHL+CLL are present in Tables 3-2 and 3-3. [Note that no trend analyses were

performed for most of these models.]

With respect to cumulative exposure to styrene and all leukemias combined, the single-

chemical (styrene) model, adjusted for age and years since hire, showed an increased risk

with increasing exposure; however, the only statistically significant risk estimate was for

the highest quartile of exposure (see Table 3-2). When butadiene was added to this

model, the exposure response was attenuated (all non-significant), and when both

butadiene and DMDTC were included in the model with styrene RRs were less than one.

The risk of leukemia increased with increasing exposure to butadiene in single-chemical,

two-chemical and three-chemical models although was somewhat attenuated after

adjusting for styrene and/or DMDTC. Signficant risk estimates for leukemia were also

observed for DMDTC, which were somewhat attenuated in two- and three-chemical


9/29/08 123

models but still remained significant. When these analyses were repeated incorporating a

ten-year exposure lag, the results were similar (Delzell et al. 2006).

1

2

3

4

5

6

7

8

These analyses were also repeated using total styrene peaks (> 50 ppm) and total

butadiene peaks (> 100 ppm). The single-chemical (styrene) model showed an increasing

trend with exposure compared with zero exposure (See Table 3-4). Intermediate and

three-chemical models also showed an increasing trend with styrene exposure, but in

each model the level of risk was somewhat attenuated (Graff et al. 2005, Delzell et al.

2006).

Table 3-2. Risk of leukemia with cumulative and peak exposurea to styrene, butadiene, and DMDTCb

Styrene exposure

No. of cases/person years

Styrene only RR (95% CI)

Styrene + butadiene RR (95% CI)

Styrene + DMDTC RR (95% CI)

Styrene + butadiene + DMDTC RR (95% CI)

Cumulative exposure, ppm-years 0 7/77,460 1.0 1.0 1.0 1.0 > 0 to < 8.3 18/177,551 1.3 (0.6–3.2) 1.2 (0.4–3.7) 0.7 (0.3–2.0) 0.6 (0.2–2.2) 8.3 to < 31.8 19/132,311 1.6 (0.7–3.9) 1.4 (0.4–4.5) 0.7 (0.3–2.1) 0.7 (0.2–2.5) 31.8 to < 61.1 18/55,797 3.0 (1.2–7.1) 1.9 (0.

6–6.5) 1.2 (0.4–3.5) 0.8 (0.2–3.1)

61.1 + 19/57,056 2.7 (1.1–6.4) 1.3 (0.4–4.3) 1.0 (0.3–2.9) 0.5 (0.1–2.0) Number of styrene peaks 0 14/202,225 1.0 1.0 1.0 1.0 > 0 to < 58 16/151,484 1.5 (0.7–3.0) 1.1 (0.4–2.9) 1.1 (0.9–3.7) 1.0 (0.4–2.7) 58 to < 170 17/53,266 3.6 (1.8–7.3) 2.6 (1.0–7.0) 2.3 (1.0–4.5) 2.0 (0.7–5.6) 170 to < 699 17/40,653 4.6 (2.3–9.4) 3.3 (1.2–8.9) 3.3 (1.4–6.6) 2.9 (1.0–8.2) 699 + 17/52,545 4.2 (2.0–8.6) 2.8 (1.0–7.8) 3.0 (1.4–6.4) 2.4 (0.8–6.9) Source: Delzell et al. 2006. DMDTC = dimethyldithiocarbamate. aPeak exposure was defined as total peaks > 50 ppm for styrene, as total peaks > 100 ppm for butadiene, and as cumulative exposure to DMDTC expressed in mg/cm-years (similar to analysis above). bModels were adjusted for age and years since hire.

Of special interest was Graff et al.’s analysis of leukemia mortality by cross-classified

cumulative exposure to styrene and butadiene. Styrene and butadiene exposure were each

categorized into three levels (low = no exposure plus the first quartile of exposure;

medium = the second and third quartiles of exposure; and high = the fourth quartile of

9

10

11

12


124 9/29/08

exposure). In the low-butadiene–exposure stratum (< 33.7 ppm-years), the RR for

leukemia mortality was 1.6 (95% CI = 0.7 to 3.9, 7 observed deaths) for the medium-

styrene–exposure group (8.3 to < 61.1 ppm-years), compared with the low-styrene–

exposure group (> 0 to < 8.3 ppm-years. No deaths occurred in the high-styrene–

exposure group (≥ 61.1 ppm-years).

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

The four categories defined by medium and high exposure to styrene (≥ 8.3 ppm-years)

and butadiene (≥ 33.7 ppm-years) had RRs between 1.6 and 3.5. The rate ratio for

leukemia mortality was 3.3 (95% CI = 1.6 to 6.7, 13 observed deaths) for the combined

high-styrene (≥ 61.1 ppm-years) and high-butadiene (≥ 425.0 ppm-years) exposure

category. In the middle- and high-butadiene exposure categories, the RR did not increase

with increasing styrene exposure. No increased risk of leukemia mortality was apparent

for workers exposed to butadiene above low levels (≥ 33.7 ppm-years) when styrene

exposure was low (< 8.3 ppm-years, RR = 1.2, 95% CI = 0.4 to 3.1, 5 observed deaths).

The marginal RR for styrene adjusted for butadiene was 1.5 (95% CI = 0.8 to 2.8) for 8.3

to < 61.1 ppm-years exposure, and 1.4 (95% CI = 0.6 to 3.0) for the 61.1+ ppm-years

category, and the test for trend was not significant with P = 0.65. [A discrepancy exists

between the person-years cited for this analysis of low-exposure styrene workers between

Graff et al. 2005 (155,011) and Delzell et al. 2006 (255,011), but no other differences in

the figures were identified.]

Graff et al. (2005) also presented separate relative risks for cumulative exposure to

styrene and CLL, AML, CML, and other leukemias. These models, using terciles of

exposure to styrene, butadiene, and DMDTC, were restricted to workers 40 years or older

and with at least 20 years of employment, and were adjusted for age and time since hire.

While increasing relative risks were observed for all subtypes of leukemia, except for

AML, in single-chemical models these risks were attenuated in three-chemical models.

At each level of styrene exposure, there were no significantly increased risks of these

subtypes of leukemia in either single- or three-chemical models, but the number of deaths

in each stratum was small (data for intermediate two-chemical models were not shown in

either report).


9/29/08 125

Single-chemical, two-chemical, and three-chemical models were also reported for

cumulative exposure to styrene, butadiene, and DMDTC for the other

lymphohematopoietic cancers (NHL and multiple myeloma). No results were reported for

Hodgkin’s disease, but the deaths were few (13) (Graff et al. 2005, Delzell et al. 2006).

Similar quartiles of cumulative exposure for the three chemicals were used as in the

leukemia analyses. No increased risk was suggested by the results for multiple myeloma.

Delzell et al. (2006) also presented data for CLL and NHL deaths combined, since CLL

and small B-cell NHL represent the same B-cell cancers. (Note that in 8 cases, a

diagnosis of both CLL and NHL was recorded, so a total of 75 rather than 83 deaths was

used in this analysis. Similar findings (patterns) were obtained for both NHL and

NHL+CLL (see Table 3-2). RRs were adjusted for age and time since hire, and the

models were restricted to workers 40 or more years of age). The risks of NHL+CLL

increased with increasing styrene exposure; the response appeared to be weaker for NHL

alone. (The only significant RR was for the highest category of styrene exposure and

NHL+CLL). When butadiene was added to the model (styrene + butadiene), the RRs for

both NHL and NHL+CLL were increased. The RRs in the intermediate model with

styrene and DMDTC in the model were somewhat reduced. When all three chemicals

were added to the model, the RRs were slightly attenuated compared with styrene alone,

and none were statistically significant. [No trend analyses were performed, but this would

have likely increased the power to detect risks associated with styrene exposure.]

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

Cumulative exposure to butadiene did not appear to be risk factor for NHL or

NHL+CLL. The risk of NHL or NHL+CLL was marginally increased at the two highest

doses of butadiene exposure, but were reduced to less than or equal to one after

controlling for styrene. RRs for DMDTC were non-significantly elevated in some

exposure categories, but no clear exposure-response relationships were observed.]


126 9/29/08

Table 3-3. Cumulative exposure to styrene, butdadiene and DMDTC and risk of NHL and NHL+CLL.

Styrene exposure ppm-years

No. of cases/person

years

Styrene only

RR (95% CI)

Styrene + butadiene

RR (95% CI)

Styrene + DMDTC

RR (95% CI)

Styrene + butadiene +

DMDTC RR(95% CI)

NHLa 0 6/53,165 1.0 1.0 1.0 1.0 > 0 to < 8.3 16/106,811 1.4 (0.5–3.6) 1.7 (0.5–5.6) 1.2 (0.4–3.2) 1.4 (0.4–4.9) 8.3 to < 31.8 11/88,810 1.1 (0.4–2.9) 1.8 (0.5–6.3) 0.9 (0.3–2.6) 1.3 (0.3–5.2) 31.8 to < 61.1 9/42,612 1.5 (0.5–4.2) 2.3 (0.6–8.7) 1.2 (0.4–3.8) 1.7 (0.4–7.0) 61.1 + 16/47,008 2.3 (0.9–5.9) 3.2 (0.9–11.2) 1.8 (0.6–5.5) 2.3 (0.6–9.2) NHL+CLLa,b 0 6/53,165 1.0 1.0 1.0 1.0 > 0 to < 8.3 20/106,811 1.7 (0.7–4.4) 2.2 (0.7–6.8) 1.4 (0.5–3.8) 1.9 (0.6–6.1) 8.3 to < 31.8 15/88,810 1.5 (0.6–3.8) 2.2 (0.7–7.1) 1.1 (0.4–3.1) 1.7 (0.5–6.2) 31.8 to < 61.1 13/42,612 2.2 (0.8–5.7) 2.7 (0.8–9.2) 1.5 (0.5–4.5) 2.0 (0.5–7.5) 61.1 + 21/47,008 3.0 (1.2–7.5) 3.1 (0.9–10.3) 2.0 (0.7–5.8) 2.2 (0.6–8.2) Source: Delzell et al. 2006. CLL = chronic lymphocytic leukemia, DMDTC = dimethyldithiocarbamate, NHL = non-Hodgkin’s lymphoma. a8 deaths had double diagnosis of CLL and NHL. bModels were restricted to 40+ years of age and were adjusted for age and year since hire.


Table 3-4. Epidemiologic studies of cancer risk following styrene exposure in the styrene-butadiene rubber industry, 1976–2005

Study


Study population and methods Exposure

Effects (SMR (95% CI), no. of observed deaths)a

Comments McMichael et al. 1976a

U.S.

Internal case-control comparison of a cohort of rubber workers

1964–72

Cohort

6,678 male workers at a rubber tire plant that produced styrene-butadiene rubber (SBR) and other rubbers

Case-control comparison Sample population: 22% of the study population (age-stratified random sample) Exposure (work group) age-adjusted risks calculated for each type of cancer or disease for workers exposed at least 2 yr and at least 5 yr, 1940–60

Work histories, obtained from personnel records, were used to assign workers to 16 major work groups; one group was a synthetic plant producing SBR and other synthetic rubbers (2%–3% of the sample population)

RR (99.9% CI)

Workers producing SBR and other synthetic rubbers with at least 5 years of exposure (significant findings) LH 6.2 (4.1–12.5) lymphatic leukemia 3.9 (2.6–8.0) Stomach cancer 2.2 (1.4 – 4.3)

Number of deaths for all 16 work areas (deaths specifically for the synthetic plant not reported) LH = 51 lymphatic leukemia = 14

SBR was the most prevalent rubber produced in the synthetic plant, but neoprene, nitrile, and ethylene-propylene-diene were also produced

Meinhardt et al. 1982, Meinhardt et al. 1978

U.S.

historical cohort

1943–76

avg. 19 yr

53,929 person-years

2,756 white male workers with ≥ 6 months of non-management or non-administrative employment in 2 SBR plants

Avg. employment ~10 yr

SMRs based on national rates

Average TWAs based on samples from all areas of production facilities were 0.94 ppm (0.03–6.46) and 1.99 ppm (0.05–12.3) in 2 plants

Study population was not subclassified according to styrene exposure

Total cohort all cancer 0.72 (0.54–0.93), 56 Cancers with non-significant excess mortality LH 1.32 (0.66–2.37), 11 NHL 1.65 (0.45–4.27), 4 leukemia 1.73 (0.63–3.73), 6

Study initiated in response to leukemia deaths of 2 workers

Study population included in studies by Delzell et al.

9/29/08 127


128 9/29/08

Study




Comments Lemen et al. 1990

follow-up to end of 1981 and 1982; 69,655 person years

As above

Additional cancer deaths (N = 50): increases reported for lung, trachea, and bronchus (27), lymphosarcoma and reticulosarcoma (2), and other LH (2), including 1 leukemia and aleukemia death. No other cancer deaths reported, and no SMRs reported.

Matanoski et al. 1997, Matanoski et al. 1993, Matanoski et al. 1990, Matanoski and Schwartz 1987

U.S. and Canada

historical cohort 1943–82

avg. 21 yr

251,431 person-years

12,110–13,920b male workers employed > 1 yr in 7 U.S. SBR plants and > 10 yr in 1 Canadian SBR plant, 1943–76


Exposure assessed from job titles; work areas obtained from personnel records

Job descriptions and tasks information obtained from plant

Total cohort all cancer 0.85 (0.78–0.93), 518 Cancers with non-significant excess mortality Hodgkin’s disease 1.20 (0.52–2.37), 8 other lymphatic 1.11 (0.64–1.77), 15 Production workers Cancers with excess mortality (non-significant and significant) kidney 1.53 (0.50–3.57), 5 LH cancer 1.46 (0.88–2.27), 19 Hodgkin’s disease 1.20 (0.15–4.35), 2 leukemia 1.34 (0.53–2.76), 7 other lymphatic 2.60 (1.19–4.94), 9

Most of the study population included in studies by Delzell et al.

Santos-Burgoa et al. 1992

U.S. and Canada

nested case-control study of LH malignancies

Cohort: Matanoski and Schwartz 1987, Matanoski et al. 1990

Cases: 59 workers who died of LH malignancies

Controls: 193 workers from cohort who were alive or had died of non-cancer causes; matched to cases by

JEM created by experts based on job titles and descriptions

Workers classified according to ranks for relative exposure to styrene and 1,3-butadiene

Cumulative exposure calculated based on exposure

OR (95% CI)

Cumulative styrene exposure > average Matched analysis LH 1.91 (0.91–4.02) leukemia 3.13 (0.84–11.2) other lymphatic 1.35 (0.25–7.40) lymphosarcoma 1.33 (0.11–16.6) Hodgkin’s disease 0.40 (0.05–3.25)

Styrene exposure ranks correlated poorly with measurements of styrene

Styrene exposure and butadiene exposure were positively


9/29/08 129

Study




Comments plant, age, year of hire, employment duration, and survival

ORs calculated by unconditional and conditional logistic regression (for matched subjects)

duration Hodgkin’s disease 0.40 (0.05–3.25) Unmatched analysis LH 1.89 (0.87–4.09) leukemia 4.26 (1.02–17.8) other lymphoma 2.42 (0.05–11.6) lymphosarcoma 1.39 (0.13–15.3) Hodgkin’s disease 0.75 (0.14–3.92) In models that controlled for butadiene exposure, risk of leukemia was not increased No indication of a positive interaction between styrene and butadiene for all LH

correlated

Matanoski et al. 1997

nested case-control study of LH malignancies

Cohort: Matanoski and Schwartz 1987, Matanoski et al. 1990

Cases: 58 workers who died of LH cancer (starting with the same 59 cases as Santos-Burgoa et al.)

Controls: 1,242 workers from cohort selected to represent distribution across plants and with similar age distribution to cases

ORs calculated with unconditional regression models (controls, N = 1,242); multivariate models included birth year, hire date, and employment duration

Mean styrene exposure for all plants = 3.53 ppm (SD = 14.32), based on 3,649 measurements (1978–83) (Matanoski et al. 1993)

Plant means = 0.29–6.66 ppm

Styrene levels averaged across jobs and plants

Average and cumulative exposure calculated from plant info., job title, and exposure duration

OR (95% CI), no. of observed deaths

Logistic regression analysis controlling for butadiene exposure risk Increase of 1 ppm in TWA styrene exposure (significant associations) LH 2.20 (1.46–3.33), 58 lymphoma 2.67 (1.22–5.84), 12 lymphosarcoma 3.88 (1.57–9.59), 7 myeloma 3.04 (1.33–6.96), 10

Cumulative exposure to styrene Increasing risks with increasing exposure LH P = 0.000c leukemia P = 0.006 myeloma P = 0.023

See above


130 9/29/08

Study




Comments Analysis done to avoid overmatching on exposure (i.e., by plant), because exposure levels differed between plants.

Delzell et al. 1996, Sathiakumar et al. 1998

Sathiakumar et al. 2005, extended follow-up

Delzell et al. 2006

U.S. and Canada

historical cohort

1943 to 1/1/1991

follow-up through 1/1/1992

avg. 25 yr


extended follow-up: 1944–1998 avg. 30 yr 540,586 person-years

Original cohort established by Delzell and colleagues 17,964 male workers employed ≥ 1 yr in 8 SBR plants, starting 1943–65 (depending on plant), followed through 1991

40 workers excluded from the extended follow up

Mortality analysis: 15,649 workers (excluding those who had not worked in SBR or related activities)

44% worked ≥ 10 yr: avg. 7.8 yr


Workers categorized from job title and department into 308 job groups organized into 5 main process groups: production (37%), maintenance (24%), labor (15%), laboratories (9%), and other operations (15%)

Workers in the subgroups polymerization (production) and maintenance (labor) had high exposure to both styrene and butadiene

Laboratory workers had high exposure to butadiene and low to moderate exposure to styrene

Workers in the subgroup coagulation (production) had low to moderate exposure to styrene but only background exposure to butadiene

Extended follow-up based on identical exposure characterization as the initial

Follow-up 1943–91: Cancers with significantly decreased mortality (total cohort) all cancer 0.93 (0.87–0.99), 950 buccal cavity & pharynx 0.50 (0.28–0.82), 15 esophagus 0.59 (0.35–0.93), 18

Leukemia total cohort 1.31 (0.97–1.74), 48 ever hourly 1.43 (1.04–1.91), 45 ever hourly (employed ≥ 10 yr, latency > 20 yr 2.24 (1.49–3.23), 28 production (job groups):

polymerization 2.51 (1.40–4.14), 15 coagulation 2.48 (1.00–5.11), 7

labor (job groups): maintenance 2.65 (1.41–4.53), 13 laboratories 4.31 (2.07–7.93), 10

Other cancers with significant excess mortality in certain subgroups: large intestine: black hourly workers with ≥ 10 yr worked and ≥ 20 yr latency lung: maintenance job group

Follow-up 1944-98: Cancers with significantly decreased

Includes workers from Meinhardt et al. 1982 (2e plants) and Matanoski et al. 1990 (7 of 8 plants)

Mortality analysis of this cohort (or subpopulations) also published by Macaluso et al. 1996, Delzell et al. 2001, and Graff et al. 2005.


9/29/08 131

Study




Comments studies. mortality (total cohort)

all cancer 0.92 (0.88–0.97), 1,608 buccal cavity & pharynx 0.47 (0.29–0.71), 22

Leukemia total cohort 1.16 (0.91–1.47), 71 ever hourly 1.23 (0.94–1.57), 63 ever hourly (employed ≥ 10 yr, latency 20–29 yr 2.58 (1.56–4.03), 19 production (job groups)

polymerization 2.04 (1.21–3.22), 18 coagulation 2.31 (1.11–4.25), 10

labor (job groups) maintenance 2.03 (1.14–3.35), 15f laboratories 3.26 (1.78–5.46), 14

Cell-type specific leukemia (ever hourly employed) CML 2.00 (1.00–3.58), 11 CLL 1.71 (0.96–2.81), 15

Significant associations also seen for CLL in polymerization, coagulation, finishing, and laboratories, for CML in laboratories and borderline significance seen for AML in maintenance labor

NHL+CLL (significant or borderline significant increased SMRs)

ever hourly 1.30 (0.99–1.67) ever hourly (employed ≥ 10 yr) by latency yr

20–29 1.90 (1.01–3.25) ≥ 30 1.49 (1.02–2.10)


132 9/29/08

Study




Comments

Job groups production (total) 1.73 (1.19–2.44) polymerization 2.18 (1.31–3.41) finishing 1.91 (1.21–2.86)


historical cohort

1943–92

418,846 person years

Cohort established by Delzell et al. 1996

Mortality analysis 16,610 workers at 6 of 8 plants (with specific work histories), including workers involved in SBR-unrelated activities

External analysis SMRs based on national rates 51 deaths from leukemia

Internal analysis RRs that adjusted for multiple exposures were computed by the Mantel-Haenszel method or by Poisson regression models 58 deaths from leukemia, including 7 defined by contributory cause of death

Plant-specific JEMs: exposure values estimated from process descriptions and surveys; TWA values linked to workers by job group

Estimated median cumulative styrene exposure was 7.4 ppm-years for 83% of the workers

Average styrene exposure decreased from 1.8 ppm in the 1940s to 0.1 ppm in the early 1990s

Leukemia

SMR (external analysis) and RR (adjusted for race, age, and cumulative exposure to butadiene) for cumulative exposure to styrene ppm-years SMR RR 0 0.89 1.0 < 5 0.63 0.9 5–9 1.61 5.4 10–39 1.36 3.4 ≥ 40 2.35 2.7

Internal analysis test for trend in RR, P = 0.14

Quantitative exposure estimates based on experts’ judgment of complex data sources; no thorough validation

Exposure estimates significantly higher than documented by measurements

Exposures highly correlated; impossible to disentangle separate styrene or butadiene effects


9/29/08 133

Study




Comments Delzell et al. 2001

historical cohort

1943–91


Mortality analysis 13,130 workers from 6 of 8 plants (similar to Macaluso et al. 1996), but also excluding 3,468 workers who died or were lost to follow-up before age 40 or 10 years latency and 12 workers with duplicate records

RRs calculated with Poisson regression models for exposure to single and multiple agents; models included age and latency

59 deaths from leukemia (1 from myelodysplasia with medical records indicating acute unspecified leukemia, in addition to 58 deaths identified by Maculuso et al. 1996

Exposure estimates by Macaluso et al. 1996 were revised (original estimates were noted as being controversial), and exposure to DMDTC was estimated

Estimated median cumulative styrene exposure for exposed workers (85%) was 17–18 ppm-years

RR (95% CI), no. of observed deaths

Leukemia Styrene exposure (ppm-years) Styrene only 0 1.0, 5 > 0–< 20.6 1.2 (0.5–3.3), 18 20.6–< 60.4 2.3 (0.9–6.2), 18 ≥ 60.4 3.2 (1.2–8.8), 18 Styrene + butadiene 0 1.0 > 0–< 20.6 1.1 (0.3–4.0) 20.6–< 60.4 1.6 (0.4–6.4) ≥ 60.4 1.8 (0.4–7.3) Styrene + butadiene + DMDTC 0 1.0 > 0–< 20.6 0.6 (0.1–2.5) 20.6–< 60.4 0.8 (0.2–3.7) ≥ 60.4 0.8 (0.2–3.8)

Revised exposure assessment gave styrene exposure levels twice as high as originally reported

See also comments for Macaluso et al. 1996

Graff et al. 2005; Delzell et al. 2006

historical cohort

1943–98



Mortality analysis: 16,579g workers at 6 of 8 plants (similar to Maculuso et al. 1996)

RR calculated by Poisson

Revised exposure estimates by Macaluso et al. 2004 were used

Individuals assigned to four quartiles of cumulative exposure to styrene (ppm-years):

Internal RR analyses: RR (95% CI), no. of deaths

Models evaluating RR for 4 categories of cumulative styrene exposure: (1) styrene only, (2) styrene + butadiene, (3) styrene + DMDTC and (4) styrene + butadiene + DMDTC (adjusted for age and time since

All 3 exposures were correlated

Spearman rank correlation with styrene exposure:


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Study




Comments regression models for exposure to single agents and exposure to multiple agents; models included age, latency, period of employment (year range), and race

SMRs based on U.S. or Ontario rates for expected numbers of LH cancer deaths

81 deaths from leukemia, 58 from NHL, 27 from multiple myeloma, and 13 from Hodgkin’s disease

0–< 8.3 8.3–< 31.8 31.8–< 61.1 ≥ 61.1

Three exposure categories were created for 1) cross-classified combined analyses; 2) RR models of CLL, AML, CML, and other leukemias, and 3) SMR analyses of AML, CML, CLL and NHL+CLL (all other analyses used quartiles): low: no exposure + 1st quartile medium: 2nd + 3rd quartiles high: 4th quartile

hire) – See data in Table 3-2 and 3-3 for all leukemia, NHL and NHL+CLL.

CLL alone by terciles of styrene exposure showed RRs of 1.0 (reference), 1.7 and 2.6 for styrene alone, and RRs of 1.0 (reference), 1.2 and 0.9 for 3-chemical model (all non-significant)

No statistically significant increases in RR were reported for CLL, AML, CML, other leukemias, or multiple myleloma (single- and 3-chemical models

RR for all myeloid neoplasms combined was significantly increased for styrene levels 31.8 – < 61.1 ppm-years in single- (2.6, 1.2–5.5, 13 deaths) but not 3-chemical models.

Analysis of cross-classified categories of butadiene and styrene; reference group = low styrene, low butadiene RR for leukemia and styrene exposure Low-butadiene exposure:

medium 1.6 (0.7–3.9) 7 high no deaths

Medium-butadiene exposure low 1.2 (0.4–3.1), 5 medium 1.6 (0.9–2.9), 25 high 1.6 (0.6–4.0), 6

High-butadiene exposure low no deaths medium 3.5 (1.3–9.3), 5

Correlation of styrene exposure with butadiene = 0.7 and with DMDTC = 0.63


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Study




Comments high 3.3 (1.6–6.7), 13

RR for styrene (ppm-year exposure) adjusted for BD 8.3 – < 61.1 1.5 (0.8-2.8) 61.1+ 1.4 (0.6-3.0) Test for trend: P = 0.65

External analysis SMRs for all leukemias and NHL increased with increasing cumulative exposure to styrene; significant at the 3rd and 4th quartiles for leukemia (1.87, 1.02–3.13, 14 deaths; 1.91, 1.09–3.10, 16 deaths, respectively) and 4th quartile for NHL (1.97, 1.08–3.31, 14 deaths), CLL (3.10, 1.01–7.24, 5 deaths) and NHL+CLL (2.29, 1.36–3.62, 18 deaths)

AML = acute myelogenous leukemia; BD = butadiene; CI = confidence interval; CLL = chronic lymphocytic leukemia; CML = chronic myelogenous leukemia; DMDTC = dimethyldithiocarbamate; IRR = incidence rate ratio; LH = lymphohematopoietic cancer; MM = multiple myeloma; NHL = non-Hodgkin’s lymphoma; OR = odds ratio; RR = relative risk or rate; SIR = standard incidence ratio; SMR = standard mortality ratio. aUnless otherwise stated. b Number of workers varied among the publications. cP-value as reported in paper. dDelzell et al. (Delzell et al. 1996) reported mortality from LH cancer, whereas Sathiakumar et al. (1998) reported mortality from all cancer. eDelzell et al. (1996) referred to these two plants as one facility. fReported in abstract of Sathiakumar et al. (2005) as SMR = 326, 95% CI = 178 to 456, which is the result for “Laboratories” in the same analysis reported in the body of the paper. gCombines the results for 16 men who had worked in different plants and had separate records and eliminates 8 men who had worked < 1 year.


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3.3 The styrene monomer and polymer industry 1

Styrene exposure levels in the styrene monomer and polymer production industries are

generally much lower than levels in the reinforced-plastics industry, and are similar in

magnitude to levels seen in the styrene-butadiene rubber production industry (see Section

2.6). Although levels exceeding 20 ppm have been reported in polymerization,

manufacturing, and purification areas for this industry, the styrene levels in maintenance,

laboratory, and packaging operations were generally less than 5 ppm. Workers in the

styrene monomer industry can also be exposed to benzene, toluene, ethylbenzene, and

other alkylbenzene compounds. In addition to benzene, toluene, and ethylbenzene,

workers in polystyrene production can be exposed to various solvents such as 1,2-

dichloroethane, carbon tetrachloride, ethyl chloride, methylene dichloride, and

chlorobenzene. Workers could also be exposed to boron trifluoride which is the preferred

initiator for the polymerization reaction (see Section 2.2). Cancer mortality for workers in

the styrene monomer and polymer industry has been studied in workers in Germany by

Frentzel-Beyme et al. (1978), in the United States by Ott et al. (1980) (with follow-up by

Bond et al. (1992) and by Nicholson et al. (1978), and in England by Hodgson and Jones

(1985). Table 3-5 provides an overview of the studies conducted in the styrene monomer

and polymer industry.

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31

3.3.1 Germany 19 Frentzel-Beyme et al. (1978) studied 1,960 workers (sex not reported) engaged in the

manufacture of styrene and styrene polymer for more than 1 month during the period

1931 to 1976. The population was identified from plant records and followed from 1956

through 1976. Percentage follow-up was much lower for non-German employees (29%),

many of whom returned to their home countries, compared with German employees

(93%). Observed numbers of cancer deaths were compared with the expected numbers

based on regional mortality rates. A total of 20,138 person-years were accumulated, and

the average follow-up was 10.3 years. Styrene exposure levels were generally below 1

ppm according to measurements conducted in 1975 and 1976 (Thiess and Friedheim

1978). Levels up to 6.84 ppm (styrene production) and 46.92 ppm (polystyrene

production) occasionally were recorded. No subclassification of workers was done that

allowed any assessment of cancer mortality by indicators of styrene exposure level. Only


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12 deaths due to malignant tumors were observed compared with 18.5 expected. A

statistically nonsignificant increase in mortality from pancreatic cancer was observed (2

observed deaths vs. 0.7 expected; P = 0.16), and mortality from lung cancer was

decreased (3 observed deaths vs. 5.4 expected; P value not reported). Non-significant

increases in mortality from rectal, peritoneal, and splenic cancer were also observed, but

these increases were based on only one observed case for each site.

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3.3.2 United States- multi-plant 7 Ott et al. (1980) studied 2,904 male workers employed for at least 1 year in the

production or research units of a company that produced styrene monomer, styrene-

butadiene latex, and styrene-based products at several U.S. locations. The workers were

identified from annual census lists for 1937 to 1970 and followed from 1940 through

1976. Bond et al. (1992) extended follow-up to 1986 and only 0.4% were lost to follow-

up. Vital status and cause of death were assessed using the company mortality

surveillance system. Mortality was compared with expected numbers calculated from

national rates and other worker populations within the company. A total of 90,000

person-years were accumulated, and average follow-up was 31 years. Industrial

hygienists assigned all manufacturing jobs (categorized into 57 groups with common

exposures) an exposure intensity with respect to five chemical exposures: (1) styrene and

ethylbenzene (1 to 4 ppm, or ≥ 5ppm), (2) benzene, alkylbenzene compounds (≥ 1 ppm),

(3) styrene, ethylbenzene, and acrylonitrile in equal concentrations (1 to 4 ppm, or ≥ 5

ppm), (4) extrusion fumes, and (5) colorants (indirect and direct exposure).

For the total study population, overall cancer mortality was significantly decreased

(SMR = 0.81, 95% CI =0. 69 to 0.95, 162 observed deaths) (Bond et al. 1992). Increased

(but statistically nonsignificant) SMRs were seen for all lymphatic and hematopoietic

malignancies (SMR = 1.44, 95% CI = 0.95 to 2.08, 28 observed deaths), Hodgkin’s

disease (SMR = 2.22, 95% CI = 0.71 to 5.18, 5 observed deaths), NHL (SMR = 1.17,

95% CI = 0.47 to 2.40, 7 observed deaths), multiple myeloma (SMR = 1.84, 95% CI =

0.74 to 3.80, 7 observed deaths), and leukemia (SMR = 1.18, 95% CI = 0.54 to 2.24, 9

observed deaths). Among workers exposed to styrene and ethylbenzene, there were 16

deaths due to lymphohematopoietic malignancies, compared with 8.1 expected [SMR =


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1.98], and mortality was slightly higher in workers exposed at least 1 year (< 1 year, 4

observed vs. 2.6 expected [SMR = 1.54]; ≥ 1 year, 12 observed vs. 5.5 expected [SMR =

2.2]) and in workers exposed to lower styrene levels (< 5 ppm, 12 observed vs. 5.1

expected [SMR = 2.4]; ≥ 5 ppm, 4 observed vs. 3.0 expected [SMR = 1.3]). A

statistically significant increased risk was found for an analysis that allowed for a 15-year

latency period (SMR = 1.60, 95% CI = 1.02 to 2.38, 24 observed deaths), but there was

no significant trend of increasing risk with increasing time since first exposure.

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3.3.3 United States- single plant 8 Nicholson et al. (1978) identified 560 male workers at a plant manufacturing styrene

monomer and polystyrene employed for at least 5 years as of 1960 according to the local

union’s seniority list. The population was followed through 1975. Cause of death (N =

83) was determined by death certificate; autopsy results were available for 18 cases and

other clinical information was available for 13 cases, and mortality was compared with

that of the general U.S. population.

NIOSH conducted a health hazard evaluation in 1974 in the plant that showed styrene

exposure levels of below 1 ppm in low-exposure areas (service and utilities) and 5 to 20

ppm in high-exposure areas (styrene production, polystyrene polymerization and

extrusion, development, and special products and maintenance). Crude styrene monomer

is produced from ethylbenzene and iron oxide, and styrene is purified by the removal of

unreacted ethylbenzene, benzene, toluene, and xylene. In addition to polystyrene

production, the plant also produced butadiene-styrene latex. The authors stated that some

individuals might have experienced high exposure to benzene during the period of 1943

to 1962.

A total of 17 workers died of any cancer (21.01 expected [SMR = 0.81]). Observed vs.

expected deaths were 6 vs. 6.99 [SMR = 0.86] for lung cancer, 1 vs. 0.79 [SMR = 1.27]

for leukemia, and 1 vs. 1.25 [SMR = 0.80] for lymphoma. In addition to the leukemia that

was the cause of death, a second individual who died from coronary disease also had a

leukemia at the time of death. The authors also reported on a review of 361 randomly

selected death certificates of individuals employed for at least 6 months (who were not

included in the cohort because they did not have 5 years of experience by 1960). The


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death certificates were obtained from either union records or as part of a company-

initiated study on its progress to NIOSH. An additional 5 leukemias and 4 lymphomas

were identified; however, information on work histories or exposures was not available.

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3.3.4 United Kingdom 4 Hodgson and Jones (1985) studied 622 male workers employed for at least 1 year from

1945 to 1974 in a plant where styrene monomer was produced, polymerized, and

processed. The workers were followed through 1978, and a total of 8,654 person years

were accumulated, with an average follow-up of 13 years. An additional 3,072 male

manual workers who had no exposure to styrene but had worked at least one year at the

site were identified as a reference group. The lymphomas were confirmed by histological

assessment by three pathologists. SMRs were computed from national mortality rates,

and standard registration ratios (i.e., SIRs) were computed from regional cancer incidence

rates. No measurements of styrene exposure were available, but the authors stated that

styrene exposure levels were in general well below 100 ppm. Workers were also exposed

to acrylonitrile, and there was potential exposure to benzene, dyestuffs, and ethylene

oxide. For the total cohort, the SMRs were 0.90 for all cancer (10 observed vs. 10.9

expected), [1.19] for lung cancer (5 vs. 4.2), and [5.36] for lymphoma (3 vs. 0.56, P =

0.02); no deaths from leukemia were observed (0 vs. 0.3). No excess of deaths from

lymphoma or leukemia was observed in the unexposed cohort. The SIRs were 2.50 for all

lymphohematopoietic malignancies (4 vs. 1.6, P = 0.079), 3.75 for lymphoma (3 vs. 0.8,

P = 0.047), and [1.67] for leukemia (1 vs. 0.6). An increased incidence of larynx cancer

was also reported (3 observed vs. 0.5 expected; P values not given); however no deaths

from larynx cancer were reported. The authors stated that laryngeal cancer is often

amenable to treatment.


Table 3-5. Cohort studies of cancer risk following styrene exposure in the styrene monomer and polymer industry, 1978–1992 Study Population, follow-up, and methods Exposure Effects Comments Frentzel-Beyme et al. 1978

Germany

1,960 workers engaged in styrene or polystyrene manufacture > 1 mo 1931–76

1956–76; avg. 10.3 yr

20,138 person-years

12 deaths from cancer

SMRs based on German city and administrative region

Date of first exposure and date of leaving plant obtained from plant records; safety precautions improved over time

Styrene exposure levels generally < 1 ppm but higher levels (up to 47 ppm) were occasionally reported (Thiess and Friedheim 1978)

SMRa (95% CI), no. of observed deaths

Cancers with > 1 death pancreas [2.77 (0.34–10.03)], 2 lung [0.55 (0.11–1.62)], 3

Incomplete follow-up for non-German workers

Low statistical power

Ott et al. 1980 Bond et al. 1992

U.S.

2,904 male workers employed ≥ 1 yr in production or research units of 1 company (several locations) that produced styrene monomer, styrene-butadiene latex, and styrene-based products; workers identified from annual census lists, 1937–70

1940–86; avg. 31 yr

90,000 person-years


RRs calculated by Mantel-Haenszel methods for cohort studies, with unexposed industrial populations within the company as reference group

Industrial hygienists assigned all manufacturing jobs (categorized into 57 groups with common exposures) an exposure intensity with respect to 5 chemical exposures: (1) styrene and ethylbenzene

(1–4 or ≥ 5 ppm) (2) benzene, alkylbenzene

compounds (≥ 1 ppm) (3) styrene, ethylbenzene,

and acrylonitrile in equal concentrations (1–4 or ≥ 5 ppm)

(4) extrusion fumes (5) colorants (indirect and

direct exposure)

SMR (95% CI), no. of deaths

Total cohort all cancer 0.81 (0.69–0.95), 162 Cancers with non-significant excess mortality LH 1.44 (0.95–2.08), 28 Hodgkin’s disease 2.22 (0.71–5.18), 5 multiple myeloma 1.84 (0.74–3.80), 7 NHL 1.17 (0.47–2.40), 7 leukemia 1.18 (0.54–2.24), 9 stomach 1.27 (0.64–2.28), 11

Non-significant increases in RRs observed for NHL, leukemia+aleukemia, and Hodgkin’s disease, and a statistically significant increase in RR for multiple myeloma (RR = 2.45, 1.07–5.65, 7 deaths) when unexposed workers were the reference group (RR for stomach cancer not reported)

Chemical grouping (obs. vs. exp.), LH cancer styrene & ethylbenzene 16 vs. 8.1

Complex mixture of exposures

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69/29/08 141

Study Population, follow-up, and methods Exposure Effects Comments Nicholson et al. 1978

U.S.

560 male workers at a styrene monomer and polymer plant employed ≥ 5 yr as of 1960 according to the local union’s seniority list

1960–75

Expected numbers of deaths based on national rates

Departments categorized into high and low exposure based on air concentrations, worker descriptions, and body burdens of metabolites measured in clinical study

Air measurements in 1974 low exposure: < 1 ppm high exposure: 5–20 ppm

Observed vs. expected deaths Total cohort all cancer 17 vs. 21.01 lung 6 vs. 6.99 leukemia 1 vs. 0.79 lymphoma 1 vs. 1.25

High and low exposure areas Data not given for specific cancers, because of small numbers


Complex mixture of exposures, including ethylbenzene, toluene, xylene, and benzene

Hodgson and Jones 1985

U.K.

622 male manual workers engaged in production of styrene monomer, polymerization, manufacture of finished products, or working in laboratory ≥ 1 yr at 1 site, 1945–74

Mortality: 1945–78, avg. 13 yr 8,654 person-years

Incidence: 1962–81

SMRs based on national rates SIRs based on regional rates

No styrene exposure measurements available; however, authors stated that styrene exposure levels were generally well below 100 ppm

SMR (obs. vs. exp. deaths) all cancer 0.90 (10 vs. 10.9) lung [1.19] (5 vs. 4.2) lymphoma [5.36] (3 vs. 0.56)* leukemia – (0 vs. 0.3)

SRR (obs. vs. exp. cases) LH 2.50 (4 vs. 1.6) lymphoma 3.75 (3 vs. 0.8)* leukemia 1.67 (1 vs. 0.6) larynx 6.0 (3 vs. 0.5), P = 0.041


Mixed exposures

CI = confidence interval, LHC = lymphohematopoietic cancer, NHL = non-Hodgkin’s lymphoma, RR = relative risk or rate, SMR = standard mortality ratio, SRR = standard registration ratio. * P (one sided) < 0.05

aCalculated from the original data using expected deaths from the Rhinehessia-Palatinate region.


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3.4 Other cohort studies 1

Other cohort studies of styrene exposure are summarized in Table 3.5. 2

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3.4.1 Styrene-exposed workers (biomarker study) 3

Anttila et al. identified 2,580 workers (distribution by sex was not reported for the

styrene cohort) biomonitored for styrene exposure through measurement of mandelic acid

in post-shift urinary samples (Anttila et al. 1998). They were followed from the first

recorded measurement made between 1973 and 1983, through 1992. A total of 34,288

person-years were accumulated [with an average follow-up of 13.3 years]. The median

mandelic acid level was 2.3 mmol/L [350 mg/L] (the authors noted that 2.9 mmol/L

urinary mandelic acid corresponded roughly to 20 ppm). (Levels were higher in women

than men, which the authors stated was probably due to the selection of the monitored

task.) Cases of cancer were identified from the Finnish Cancer registry, and SIRs were

computed from expected values estimated from cancer incidence rates in the general

population. The overall cancer incidence was decreased (SIR = 0.80, 95% CI = 0.59 to

1.06, 48 observed cases), and the incidence of rectal cancer was significantly increased

(SIR = 3.11, 95% CI = 1.14 to 6.77, 6 observed cases). Increased risks were indicated for

cancer of stomach, liver, pancreas, and nervous system and Hodgkin’s disease, but none

of the findings were statistically significant. When the analysis was limited to workers

followed for at least 10 years after first measurement, the SIR was 3.49 (95% CI = 0.72 to

10.2, 3 observed cases) for rectal cancer, 3.54 (95% CI = 0.09 to 19.7, 1 observed case)

for liver cancer, 3.64 (95% CI = 0.75 to 10.6, 3 observed cases) for pancreatic cancer, and

3.11 (95% CI = 0.85 to 7.95, 4 observed cases) for cancer of the nervous system; no cases

of lymphohematopoietic malignancy were observed. SIRs were not higher in the high-

exposure group (based on lifetime mean urinary metabolite levels) compared with the

low-exposure group, but the numbers of observed and expected cases were low. The

authors did not provide sex-specific risk estimates but stated that there was no clear

difference in the overall incidence pattern between styrene-exposed men and women.

3.4.2 Environmental exposure 28

Loughlin et al. (1999) evaluated the mortality from lymphatic and hematopoietic

malignancies among 15,403 students (7,882 men and 7,521 women) attending a high


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school adjacent to a styrene-butadiene rubber plant between 1963 and 1993 for at least

three consecutive months during a school year. The population was identified from high

school yearbooks, and school records (which do not allow recording of sex). Sex was

assigned based on data from birth records (73%) of the population, yearbook pictures,

and student’s name. Data on name changes (such as married name) that occurred after

high school were obtained from multiple searches of marriage databases (the first round

used the “maiden name,” and date of birth, and the second round used the “married

name”). The population was followed through 1995, and vital status was obtained from

the National Death Index, the Social Security Administration Death Master Files, and the

Texas Department of Health death database. Cause of death was obtained from death

certificates (matching on maiden or married name, date of birth and state of birth), and

SMRs were based on expected numbers calculated from national death rates. A

statistically nonsignificant increase in overall cancer mortality was observed for men

(SMR = 1.22, 95% CI = 0.83 to 1.73, 31 deaths) but a significant decrease was observed

for women (SMR = 0.52, 95% CI = 0.28 to 0.88, 13 deaths). The sex-specific SMRs were

as follows (men vs. women): all lymphohematopoietic malignancies, 1.64 (95% CI =

0.85 to 2.87, 12 deaths) vs. 0.47 (95% CI = 0.06 to 1.70, 2 deaths); Hodgkin’s disease,

1.46 (95% CI = 0.18 to 5.28, 2 deaths) vs. 1.20 (95% CI = 0.03 to 6.68, 1 death); and

leukemia, 1.82 (95% CI = 0.67 to 3.96, 6 deaths) vs. 0.45 (95% CI = 0.01 to 2.48, 1

death). Among males, the SMR’s for subtypes of lymphohematopoietic cancers were

somewhat higher in those who attended school for 2 years or less compared with those

who attended more than 3 years. [Note that only the SMR for leukemia+aleukemia

among those attending high school for < 2 years was significantly elevated, SMR = 5.29,

(95% CI = 1.09 to 15.46, 3 deaths)]. A significant excess of deaths from benign

neoplasms (all of which were brain tumors) also was observed in men (SMR = 6.27, 95%

CI = 2.04 to 14.63, 5 deaths); only one case was observed in females (SMR = 1.56, 95%

CI = 0.04 to 8.71, 1 death).

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Table 3-6. Other cohort studies evaluating cancer risk and exposure to styrene

Study Population, follow-up, and

methods Exposure Effects Comments Anttila et al. 1998

Finland

2,580 workers biologically monitored for styrene exposure, starting 1973–83 and followed through 1992

[avg. 13.3 yr]

34,288 person-years (styrene)

SIRs based on national rates

Exposure assessed by measuring post-shift MA concentration in urine

median = 2.3 mmol/L range = 0–47 mmol/L

Median urinary MA level corresponds to a styrene concentration in air of about 20 ppm

SIR (95% CI), no. of observed cases Total cohort all cancer 0.80 (0.59–1.06), 48 Cancers with significantly or nonsignificantly increased incidences rectum 3.11 (1.14–6.77), 6 stomach 1.40 (0.45–3.26), 5 liver 1.63 (0.04–9.08), 1 pancreas 1.66 (0.34–4.85), 3 nervous system 1.61 (0.59–3.50), 6 Hodgkin’s disease 1.89 (0.23–6.84), 2 ≥ 10 years after first measurement Cancers with increased incidencesa rectum 3.49 (0.72–10.2), 3 pancreas 3.64 (0.75–10.6), 3 nervous system 3.11 (0.85–7.95), 4

Well-characterized styrene exposure

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9/29/08 145

Study Population, follow-up, and

methods Exposure Effects Comments Loughlin et al. 1999

U.S.

15,403 students (7,882 male, 7,521 female) attending a high school located near an SBR plant, 1963–93

1963–95

avg. 20.1 yr



No exposure assessment

Study prompted by potential exposure to plant emissions

SMR (95% CI), no. of observed deaths Men (241 deaths) Cancers with excess mortality (significant or non-significant all cancer 1.22 (0.83–1.73), 31 LH 1.64 (0.85–2.87), 12 Hodgkin’s disease 1.46 (0.18–5.28), 2 leukemia 1.82 (0.67–3.96), 6 other lymphopoietic 2.05 (0.56–5.26), 4 peritoneum 1.26 (0.41–2.94), 5 respiratory 1.46 (0.47–3.40), 5 benign (brain) 6.27 (2.04–14.63), 5 lower mortality among long-term students (≥ 3 yr), except for Hodgkin’s disease Women (97 deaths) all cancer 0.52 (0.28–0.88), 13 Cancers with non-sigificant excess mortality Hodgkin’s disease 1.20 (0.03–6.68), 1 benign (brain) 1.56 (0.04–8.71), 1

Questionable completeness of study population

Questionable identification of death certificates, especially among women

aOnly cancers for which the SIR was higher after ≥ 10 years than 0–9 years of follow-up and there was > 1 exposed case.


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3.5 Case-control and ecological studies 1

Four case-control studies and one ecological study in which potential exposure to styrene

was analyzed, together with a series of case-control studies among a population in

Montreal, Canada, are summarized briefly in the text that follows. Details of the study

design, sample sizes, and findings are included in Table 3-7.

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3.5.1 Lymphohematopoietic cancers 6

Flodin et al. (1986) conducted a clinic-based, case-control study in Sweden of 59 patients

diagnosed with acute myeloid leukemia between 1977 and 1982 and 354 controls. A total

of 354 population controls was selected, 236 matched (4 per case) on gender, age, and

place of residence, and 118 unmatched (2 per case). The study focused primarily on the

effect of background gamma radiation on the incidence of acute myeloid leukemia. A

self-administered questionnaire was mailed to eligible participants and included questions

on sources of radiation exposure, 10 different occupational exposures, medical care, and

lifestyle exposures. The response rate for questionnaire completion was not specified.

The method of assessing solvent and other chemical exposures from the “qualitative”

information about solvent exposure provided on the questionnaires was also not clarified.

Data were analyzed by logistic regression. The OR for 3 cases of acute myeloid leukemia

following styrene exposure (vs. 1 referent) was 18.9 (95% CI = 1.9 to 357). [Note that it

appears from the data presented that this OR is unadjusted for other potentially

confounding variables.]

Guenel et al. (2002) conducted a nested case-control study among a population of French

utility workers. Seventy-two cases of leukemia (ICD-9 204–208) among active workers

below the age of 60 and 285 controls matched by birth year were identified for the study

period of 1978 to 1989. Occupational exposures were assigned by company physicians,

toxicologists, and epidemiologists, using a job-exposure matrix (JEM) based on job title,

job tasks, and place of work. In addition, the cumulative duration (% of work time-years)

but not intensity of exposure was estimated for a group of chemicals that included

styrene. The OR (adjusted for benzene and several other chemical exposures) for

potential exposure to styrene (estimated from a JEM) was 1.1 (95% CI = 0.2 to 5.9) based

on 2 exposed cases and 9 exposed controls.


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Seidler et al. (2007) conducted a population-based, case-control study of exposure to

chlorinated and aromatic organic solvents and malignant lymphoma incidence among

men and women between 18 and 80 years of age in six regions of Germany. Cases (710)

were matched by age, region, and gender to equal numbers of population controls.

Cumulative occupational exposures were evaluated by detailed personal work histories

obtained by face-to-face questionnaire and assessment by an occupational physician;

exposure was estimated by both duration (% of work time) and 3 levels of exposure. Data

were analyzed by conditional logistic regression. In comparison with 542 cases with no

estimated exposure to styrene, the ORs for malignant lymphoma associated with styrene

exposure, after adjustment for smoking and alcohol consumption, were 0.7 (95% CI = 0.5

to 1.0, 70 cases) for > 1 to 1.5 ppm-years; 1.2 (95% CI = 0.8 to 1.7, 79 cases) for > 1.5 to

67.1 ppm-years; and 0.8 (95% CI = 0.3 to 1.4, 12 cases) for > 67.1 ppm-years. No

significant trend with exposure was observed (P = 0.43). No elevated risks were observed

when lymphoma subtypes were considered. No attempt was made in this study to adjust

for potential confounding by multiple exposures, including other aromatic hydrocarbons

and chlorinated hydrocarbons. [Note that chlorinated hydrocarbons, but not other

aromatic hydrocarbons, were associated with an elevated risk of malignant lymphoma.]

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3.5.2 Breast cancer 18

Cantor et al. (1995) conducted a population-based, case-control study of breast cancer

mortality based on records gathered between 1984 and 1989 from 24 U.S. states. Cases

were 33,509 women with breast cancer listed as the underlying cause of death on the

death certificate, and 117,794 controls were randomly selected from non-cancer deaths

matched for gender, age within 5 years, and race. Homemakers were excluded from the

analysis. A JEM was used to classify cases and controls with respect to specific

occupational exposures and the probability and level of exposure. The JEM was based on

the usual occupation and industry listed on the death certificate, from which an industrial

hygienist assigned a probability of exposure or a probable level of exposure for a total of

31 agents or groups of agents. Analyses were adjusted for age at death and, in some

cases, socioeconomic status, and results were presented separately for black and white

women. Among white women, in comparison with 27,610 cases with no estimated


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styrene exposure, the ORs for breast cancer, adjusted for age and socioeconomic status,

were 1.16 (95% CI = 1.1 to 1.3, 807 cases) for low exposure, 1.13 (95% CI = 1.0 to 1.3,

522 cases) for medium exposure and 1.19 (95% CI = 0.9 to 1.6, 70 cases) for the highest

exposure category. Among black women, in comparison with 3,918 non-exposed cases,

the adjusted ORs for low- and medium-exposure levels were 1.59 (95% CI = 1.2 to 2.1,

87 cases) and 1.41 (95% CI = 1.0 to 1.9, 63 cases), respectively. Thus, breast cancer

showed a weak but statistically significant association with styrene exposure, with ORs

generally about 1.2 for whites and 1.5 for blacks. No clear trend by exposure probability

or exposure level was seen that was consistent across the two races. [Note that no other

exposures investigated were significantly associated with breast cancer in this population,

with the exception of a weak association with asbestos and non-ionizing radiofrequency

and ionizing radiation.]

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Coyle et al. (2005) conducted an ecological study to evaluate the relationship between

invasive breast cancer incidence and releases of 12 selected environmental toxicants

reported in the paper to be associated with breast cancer (the chemicals carbon

tetrachloride, formaldehyde, methylene chloride, styrene, tetrachloroethylene, and

trichloroethylene and the metals arsenic, cadmium, chromium, cobalt, copper, and nickel)

that were reported to the Environmental Protection Agency as being released in one or

more of 254 counties in Texas during 1988 to 2000. During the years 1995 through 2000,

54,487 cases of breast cancer (in both men and women) were identified from the Texas

Cancer Registry. For each toxicant, the age-adjusted breast cancer rate for each of these

counties was compared with the amount of toxicant released in that county, based on

information obtained from the EPA Toxics Release Inventory (TRI) for 1988 to 2000. In

a univariate analysis, the median age-adjusted annual breast cancer incidence was

significantly higher in counties reporting releases of styrene (and several other

compounds) than counties not reporting releases (66.2 cases in 61 counties vs. 59.8 cases

in 193 counties, respectively, P < 0.001). A multivariate analysis model of breast cancer

and exposure to the environmental toxicants (that were significantly associated with

breast cancer in the univariate analyses) found significant positive associations between

release of styrene and breast cancer in women and men (P = 0.0004), women (P = 0.002),

and women aged 50 or older (P = 0.002). [No other data were presented, and it is not


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clear why linear regression was used.] The model adjusted for age, ethnicity, race, and

exposure to those environmental toxicants that were significantly associated with breast

cancer in univariate analyses. [The criterion for exposure (one or more releases reported

in the TRI in a given county) was unlikely to reflect individual styrene exposure. The

ecological nature of the study did not allow for the evaluation of other factors (such as

socioeconomic status) that may also differ between counties with high and low breast

cancer rates but may correlate with exposure.]

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3.5.3 Series of studies in a Canadian population 8

A series of population-based, case-control studies of occupational risk factors was

conducted according to similar protocols and within the same population in Montreal,

Canada (Dumas et al. 2000, Gérin et al. 1998, Parent et al. 2000). A total of 3,730 male

cancer patients (with cancer of the esophagus, stomach, colon, rectum, pancreas, lung,

prostate, bladder, and kidney, melanoma of the skin, NHL, and Hodgkin’s disease) were

evaluated from 1979 to 1986. As controls for each cancer site analyzed, Gérin et al. used

a sample of 533 other cancer patients pooled with a sample of 533 male population

controls; Dumas et al. used all other patients with cancer at other sites (excluding lung

cancer and anatomically contiguous cancers); and Parent et al. used cancer patients (as in

Dumas et al. 2000) pooled with the 533 population controls. Case and control subjects

were interviewed about the characteristics of each job held, and chemists and hygienists

translated each job case-by-case into potential exposure to styrene, styrene-butadiene

rubber, and some 300 other substances. Data were analyzed by unconditional logistic

regression with adjustment for age, smoking, and respondent status in all studies; body

mass index (Dumas et al. 2000, Parent et al. 2000); family income and ethnic group

(Gerin et al. 1998); and education and beer consumption (Dumas et al. 2000).

In analyses that focused on four different organic solvents, Gerin et al. (1998) found

statistically significant increased ORs with respect to exposure to medium/high levels of

styrene for rectal cancer (OR = 5.1, 95% CI = 1.4 to 19.4, 5 cases), and prostate cancer

(OR = 5.5, 95% CI = 1.4 to 21.8, 7 cases), and statistically nonsignificant increased risks

of NHL (OR = 2.0, 95% CI = 0.8 to 4.8, 8 cases), and Hodgkin’s lymphoma (OR = 2.4,

95% CI = 0.5 to 11.6, 2 cases). No increases in the risk for cancer of the esophagus,


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stomach, colon, pancreas, lung, bladder, or kidney or melanoma of the skin were

observed. [Note that these ORs were adjusted for demographic and socioeconomic

covariates, smoking, and respondent status but not for other exposures.] Only 2% of the

population was classified as exposed to styrene and only 45% of those were considered to

be “certainly” exposed. Dumas et al. (2000) focused on a broad spectrum of occupational

factors and the risk of rectal cancer and found a statistically increased unadjusted OR for

styrene exposure (for “substantial exposure,” unadjusted OR = 3.9, 95% CI = 1.2 to 12.9,

5 cases; for “any” exposure, OR adjusted for demographic and lifestyle factors = 1.7,

95% CI = 0.7 to 4.5, 6 cases. Note that no adjustment for potential confounders was

conducted for the “substantial” exposure group). Parent et al. (2000) examined the risk

factors for renal-cell carcinoma and found that the OR for exposure to styrene-butadiene

rubber was significantly increased for renal-cell carcinoma among the “any exposure”

group (OR = 2.1, 95% CI = 1.1 to 4.2, 10 cases, adjusted for demographic and lifestyle

variables, but the risk was somewhat attenuated when additionally adjusted for felt dust

exposure (OR = 1.8, 95% CI = 0.9 to 3.7).

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3.5.4 Lung cancer and styrene exposure 16

Scélo et al. (2004) conducted a clinic-based, case-control study of 2,861 patients (of a

total of 3,403) diagnosed with lung cancer between 1998 and 2002 in Romania, Hungary,

Poland, Russia, Slovakia, the Czech Republic, and the United Kingdom. There were

3,118 hospital or population controls. Cases and controls were interviewed about the

characteristics of each job held, and chemists and hygienists translated each job case-by-

case into potential exposure to styrene and 70 other agents. The OR for lung cancer

among patients ever exposed to styrene (N = 51) was 0.7 (95% CI = 0.42 to 1.18;

adjusted for center, gender, age, smoking, vinyl chloride, acrylonitrile, formaldehyde, and

inorganic pigments). No trends with duration of exposure or cumulative exposure were

observed.


Table 3-7. Case-control and ecological studies evaluating cancer risk and exposure to styrene Study Population and methods Exposure Effects Comments Flodin et al. 1986

Sweden

clinic-based case-control study

Cases: 59 Swedish patients (aged 20–70) diagnosed with acute myeloid leukemia at medical clinics or departments in hospitals, 1977–82

Controls: 354 Swedish referents: 236 matched controls (4 per case) selected from general population register and matched for gender, age, and locality; 118 randomized controls (2/case) from the general population register of the hospital catchment area, aged 20–70

ORs calculated by logistic regression

Exposure to styrene and other agents (radiation, solvents), and information on smoking and other lifestyle exposures assessed by questionnaire

OR (95% CI), cases/controls (unadjusted)

Styrene exposure acute myeloid leukemia 18.9 (1.9–357), 3/1

Self-reported exposure information obtained after diagnosis was made

Only 3 styrene-exposed cases

Guenel et al. 2002

France

nested case-control study of active utility workers

Cases: 72 cases of leukemia diagnosed 1978–89 (< 60 years old)

Controls: 285 controls (4 per case) matched on birth year

ORs calculated by conditional logistic regression

Exposure to styrene and other agents (electromagnetic fields, radiation, other solvents, asbestos, etc.) assessed by company by job title, type and place of work using a JEM

OR (95% CI), no. of cases/controls Styrene exposure Leukemia OR (adjusted for benzene + other chemicals) 1.1 (0.2–5.9), 2/9

Cantor et al. 1995

U.S.

population-based, case-control study

Cases: 33,509 women with breast cancer as underlying cause of death (1984–89) in a database of 24 states; homemakers excluded, leaving 29,397 white women and 4,112 black women

Controls: 117,794 controls (4/case)

Occupation title and industry obtained from death certificates; JEM linked this information with occupational hygiene literature to estimate the probability (4 levels) and level (3 levels) of exposure to 31 specific occupational

adjusted OR (95% CI), no. of cases adjusted for age at death and socioeconomic status Styrene exposure and breast cancer

White women probability

1 1.13 (1.0–1.2), 804 2 1.18 (1.1–1.3), 527

Styrene exposure based only on “usual occupation” on death certificate

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Study Population and methods Exposure Effects Comments randomly selected from noncancer deaths; frequency matched for age (5-yr groups), gender, and race; homemakers excluded, leaving 102,955 white women and 14,839 black women

ORs adjusted for age at death and socioeconomic status

agents, including styrene 3 1.38 (1.0–1.9), 64 4 NR

level 1 1.16 (1.1–1.3), 807 2 1.13 (1.0–1.3), 522 3 1.19 (0.9–1.6), 70

Black women probability

1 1.49 (1.1–2.0), 80 2 1.52 (1.1–2.1), 61 3 1.32 (0.5–3.3), 7 4 NR

level 1 1.59 (1.2–2.1), 87 2 1.41 (1.0–1.9), 63 3 NR

Coyle et al. 2005

Texas, USA

ecological study

54,487 cases of invasive breast cancer in men and women reported to the Texas Cancer Registry, 1995–2000 Age-adjusted breast cancer rates for each of the 254 Texas counties compared with the amount of toxicant (for 12 toxicants) released in those counties

Univariate analysis: Mann-Whitney U test used to compare median average annual age-adjusted breast cancer rates in counties reporting releases vs. those not reporting releases Stepwise multiple linear regression models included age, race, ethnicity, and toxicants

The amount of toxicant released in each county in 1988–2000 obtained from the EPA Toxics Release Inventory. Release information obtained for 12 chemicals or metals: carbon tetrachloride, formaldehyde, methylene chloride, styrene, tetrachloroethylene, trichloroethylene, arsenic, cadmium, chromium, cobalt, copper, and nickel.

Toxicants were chosen based on (1) reporting of an association with breast cancer

Univariate analysis median average annual age-adjusted breast cancer rate in Texas counties Reported release of styrene yes 66.2 cases no 59.8 cases, P < 0.001

Multiple linear regression: styrene and age-adjusted breast cancer rate β, P, explained variance (%) men & women 0.219, 0.0004, 9 women 0.191, 0.002, 8 women ≥ 50 0.187, 0.002, 14 women < 50 NR, > 0.05

Criterion for exposure (≥ 1 releases reported in TRI in a given county) unlikely to reflect individual styrene exposure

Ecological nature of the study did not allow for the evaluation of other factors that may also differ between counties with high and low breast cancer rates


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Study Population and methods Exposure Effects Comments significantly associated with breast cancer in the published literature, (2)

designation by EPA as carcinogens or substances with estrogenic effects, and (3) consistent reporting of releases to TRI in 1988–2000

Unclear why linear regression analysis employed

Gérin et al. 1998a

Dumas et al. 2000

Parent et al. 2000

Canada

population-based case-control study

Cases: 3,730 men aged 35–70 with cancer identified 1979–86; cancers at 15 sites studieda Cancer no. of cases renal-cell carcinoma 142 rectal 257 prostate 449 NHL 215 Hodgkin’s disease 54

Controls: Cancer controls: patients with cancer at different sites, excluding lung and anatomically contiguous sites Population controls: 533 men selected from electoral list, age distribution similar to casesGérin et al. used the 533 population controls and a subset (533) of the cancer controls

ORs computed by unconditional logistic regression analysis with adjustment for age, respondent status, cigarette smoking (all publications), plus (1) family income, ethnic group (Gérin et al.)(2) education, beer consumption, body mass index (Dumas et al.)

Case and control subjects interviewed about characteristics of each job; chemists and hygienists translated each job into potential exposure to styrene, styrene-butadiene rubber, and 300 other substances

Styrene exposure and various cancers, using pooled (population & cancer) controls (Gérin et al.) adjusted OR (95% CI), cases/controls Cancers with increased ORs Medium or high exposure rectum 5.1 (1.4–19.4), 5/4 prostate 5.5 (1.4–21.8), 7/3 esophagus 1.4 (0.5–3.8), 5/40 Ever exposed NHL 2.0 (0.8–4.8), 8/19 Hodgkin’s disease 2.4 (0.5–11.6), 2/19

Styrene exposure and rectal cancer, using cancer controls (Dumas et al.) adjusted OR (95% CI), no. of exposed cases any 1.7 (0.7–4.5), 6 substantial 3.9 (1.2–12.9), 5

Styrene-butadiene rubber exposure and renal-cell cancer, using pooled controls (Parent et al.) adjusted OR (95 % CI), no. of exposed cases Model 1 2.1 (1.1–4.2), 10 Model 2 1.8 (0.9–3.7), 10

Styrene-exposed workers dominated by firefighters (35%), mechanics and repairmen (20%), and painters (11%), which are not generally known as groups exposed to high levels of styrene Only 2% of the population had potential styrene exposure


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Study Population and methods Exposure Effects Comments (3) body mass index (Parent et al.): Model 1 (4) body mass index and felt dust exposure (Parent et al.): Model 2

Scélo et al. 2004

Romania, Hungary, Poland, Russia, Slovakia, Czech Republic, U.K.

clinic-based, case-control study of lung cancer

Cases: 2,861 patients with newly diagnosed lung cancer that occurred 1998–2002; patients recruited from 15 hospital centers

Controls: 3,118 controls; most (at 13 of the 15 centers) were hospital controls recruited from the same hospital or area as the cases and without tobacco-related diseases; 2 centers used population controls recruited from the population or general practitioners’ registers.

ORs calculated by unconditional logistic regression and adjusted for age, gender, center, tobacco consumption, and exposure to occupational agents

Case and control subjects interviewed about characteristics of each job; chemists and hygienists translated each job into potential exposure to styrene and 70 other agents

0.6% of controls had potential exposure to styrene based on jobs held

Adjusted OR (95% CI), cases/controls

Styrene exposure and lung cancer

Ever exposed 0.70 (0.42–1.18), 51/47

(adjusted for age, gender, center, smoking, vinyl chloride, acrylonitrile, formaldehyde, organic pigments)

Risk of lung cancer did not increase with increasing duration of exposure, weighted duration of exposure, or cumulative exposure

80% statistical power to detect OR for ever exposure in the range of 1.5 to 1.6


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Study Population and methods Exposure Effects Comments Seidler et al. 2007

Germany

multi-center population-based case-control study of malignant lymphoma

Cases: 710 newly diagnosed male and female patients 18–80 years old prospectively recruited from 6 regions.

Controls: 710 controls recruited from population registers, matched on age, gender, region.

ORs calculated by conditional and unconditional logistic regression and adjusted for smoking and alcohol consumption.

Cases and controls interviewed about detailed job histories and leisure activities; exposure assessments conducted by occupational physician blind to status of participants to organic solvents including styrene, toluene, xylene and benzene, and 4 chlorinated hydrocarbons.

161 cases had estimated exposure to styrene; 542 cases had no estimated styrene exposure.

Adjusted OR (95% CI) cases/controls

ppm yrs: > 0 1.5 0.7 (0.5–1.0), 70/85 > 1.5–67.1 1.2 (0.8–1.7), 79/67 > 67.1 0.6 (0.3–1.4), 12/17 test for trend: P = 0.43

No adjustment made for potential confounding due to multiple exposures

No association found between other subtypes of lymphoma and styrene

Significant association found between chlorinated hydrocarbons and lymphoma, but not other aromatic hydrocarbons

CI = confidence interval, NHL = non-Hodgkin’s lymphoma, NR = not reported, OR = odds ratio. aGerin et al. reported results for all cancer sites, Dumas et al. reported results on rectal cancer, and Parent et al. reported results for renal cell carcinoma.


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3.6 [Strengths and limitations of the literature]3 1

This section discusses the utility of the studies for assessing the possible carcinogenicity

of styrene (3.6.1), limitations of studies due to potential misclassification (3.6.2), and

other possible sources of bias or confounding (3.6.3).

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3.6.1 Utility of the studies 5

With respect to cohort studies, the possible carcinogenicity of styrene has been assessed

among more than 100,000 workers employed in styrene-related industries. Workers in the

reinforced-plastics industry may have experienced styrene exposure levels that may be

considerably higher than workers in the styrene-butadiene rubber and styrene monomer

and polymer industries (Delzell et al. 2001, Jensen et al. 1990, Kogevinas et al. 1994b,

Macaluso et al. 1996, Macaluso et al. 2004, Thiess and Friedheim 1978). Furthermore,

the reinforced-plastics industry, unlike the two other industries, is characterized by

exposure to few other suspected carcinogens (Jensen et al. 1990). Results for workers

biomonitored for styrene are also informative for this industry, because their styrene

exposure was well characterized and because most of the workers monitored were

laminators in the reinforced-plastics industry (Anttila et al. 1998). However, this study

did not examine cancer risk by duration or level of exposure.

On the other hand, studies of the reinforced-plastics industry included few long-term

workers, with the exception of the large multi-country cohort of Kogevinas et al. (1994a,

1993). Of approximately 85,000 reinforced-plastics workers studied, the majority were

employed for less than one year, and fewer than 7,500 were employed for more than 10

years. An estimated 40% of the latter workers were laminators (the workers with the

highest styrene exposure level), so the database includes results for only 3,000 long-term

workers exposed to styrene at high levels. Secondly, the average follow-up was less than

15 years for three of the most informative populations (Anttila et al. 1998, Kogevinas et

al. 1994a, Kolstad et al. 1995). Thirdly, as in the case of the majority of other populations

exposed to styrene, exposures have been considerably reduced over the past decades (for

example, in the study by Anttila et al. (1998) exposures among laminators had been

3 The title of this section is bracketed to indicate the presence of opinion throughout this section rather than bracketing specific statements.


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reduced from approximately 200 ppm in the 1960s to less than 100 ppm by the 1970s).

Thus, workers who started employment in earlier years are likely to have had higher

exposures than those hired in recent years. Nevertheless few of the studies report any

analyses by year of first hire. In addition, none of these cohort studies (except for Antilla

et al. 1998) used quantitative measures of exposure.

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With respect to studies of styrene-butadiene rubber workers, there are a large number of

person-years of exposure among several large cohorts, which have been followed over

several decades. In addition, most plants in the styrene-butadiene rubber industry were

brought into operation at about the same time (the 1940s) and workers in them are likely

to have experienced similar patterns of decline in exposure levels over time. As in the

reinforced-plastics industry, exposure to styrene has decreased over the past several

decades. However, none of the studies have fully examined the effect of year of hire on

cancer rates, and analyses by cumulative exposure or duration of exposure do not reflect

these changes in exposure over time. The other main limitation of these cohort studies, in

addition to exposure misclassification, is co-exposure to butadiene, a known carcinogen;

this is of particular concern when analyzing lymphohematopoietic cancers.

The cohort studies of styrene monomer and polymer workers are small and lack sufficient

statistical power to detect moderate increases in risk. In addition, few cancer outcomes

have been studied, and workers in this industry are exposed to multiple chemicals,

several of which, e.g., benzene, are known or suspected carcinogens.

Overall, the statistical power of the total epidemiologic database of cohort studies is only

sufficient to detect markedly increased risks. Negative findings, even among the highly

exposed study populations, should therefore be interpreted with caution, because a

relatively small number of workers may have experienced relevant cumulative styrene

exposure and time since first exposure.

The clinic- and population-based, case-control studies provide only limited relevant

evidence, in large part because of low statistical power to detect an effect, which in turn

is due mainly to the fact that high-level styrene exposure is rare in the general population

(probably below 0.1% according to Gérin et al. (1998); only 2% of the population in his


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Montreal-based study were considered to be potentially exposed to any level of styrene).

The one occupational case-control study, of utility workers by Guenel et al. (2002), was

primarily focused on electromagnetic field exposure and yielded only 11 of 357 workers

(3%) with potential styrene exposure. In addition, the general lack of precision with

which exposures were assigned also reduced the power of these studies to detect an

effect, as discussed below. However, in the case of the large case-control study of breast

cancer (Cantor et al. 1995), this study may be of value in assessing the risk of this cancer

among the women exposed to styrene, since there is generally insufficient power to detect

breast cancer risk among women in cohort studies due to the small number of exposed

women.

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3.6.2 Misclassification of disease and exposure 11

Only three of the reviewed cohort studies used incidence data to classify health

outcomes — one report on a U.K. cohort by Hodgson and Jones (1985), two reports on a

Danish cohort by Kolstad et al. (1995, 1994), and a report on a Finnish cohort by Anttila

et al. (1998). The other cohort studies were based on mortality data, which may provide

less reliable information about diagnosis, and which may not include cases with other

causes of death or cases resulting in death after the end of the follow-up period. Among

the Danish reinforced-plastics workers, 74% of the male patients with a recorded

diagnosis of lymphohematopoietic malignancy in the national cancer registry had this

diagnosis recorded on the death certificate (Kogevinas et al. 1994b, Kolstad et al. 1994).

In the cohort of styrene-butadiene rubber workers studied by Delzell and colleagures

(Delzell et al. 2001), medical records were obtained for a subset (majority) of the workers

who died of leukemia, NHL, multiple myeloma, and Hodgkin’s lymphoma, and the

majority of the diagnoses were confirmed. One of the main methodological challenges in

the analysis by different types of lymphohematopoietic cancers, particularly when based

only on death certificate data, is the possibility of misclassification of different subtypes

of leukemia and between different types of lymphohematopoietic cancers, e.g., leukemias

and lymphomas.

Only a few studies have assessed specific sub-diagnoses of leukemia (Delzell et al. 2006,

Flodin et al. 1986, Graff et al. 2005, Kogevinas et al. 1994a, Kolstad et al. 1996,


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Sathiakumar et al. 2005). (AML, CML, and adult ALL arise from the same pluripotential

stem cell, based on observations of specific genetic re-arrangements in these 3 subtypes

of leukemia, which comprise about 80% of adult leukemias. For example, the blast crisis

of CML, 90% of which have the Philadelphia chromosome, cannot be distinguished from

AML. An estimated 10% of adult ALL cases have the Philadelphia chromosome, which

suggests a common stem-cell origin for these leukemias (Bloomfield et al. 1978, Jacobs

1989, Yunis 1983). There is considerable overlap between CLL and NHL; CLL and NHL

(85%) are B-cell malignancies (Delzell et al. 2006) and CLL is the same disease as small

lymphocytic lymphoma (Delzell et al. 2006, Harris et al. 2000). Delzell et al. 2006

grouped NHL+CLL in their data analyses.)

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The major limitation of the studies reviewed is potential misclassification of styrene

exposure and potential confounding by co-exposures. In particular, there are no ambient

air monitoring data for earlier calendar years when exposure is known to have been

considerably higher than in recent years. [Some of the analytical methods used in

exposure analyses are old; see Section 2.3.] In the smaller cohort studies (Frentzel-Beyme

et al. 1978, Hodgson and Jones 1985, Meinhardt et al. 1978, Nicholson et al. 1978), no

attempts were made to differentiate workers according to styrene exposure, and an

unknown proportion may actually have been unexposed. The same limitation also

pertains to the Danish studies of the reinforced-plastics industry; however,

misclassification may have been less, because most of the Danish workers were

employed in small companies and thus virtually all employees would have exposure to

styrene (Kolstad et al. 1995, Kolstad et al. 1994, Kolstad et al. 2005). The temporal

variation in styrene exposure levels can be another source of misclassification over the

study period (Kolstad et al. 2005, Macaluso et al. 2004). (See discussion of temporal and

job/task variation in exposure reported by Macaluso et al. 2004 and Figuer 2-4 and 2-5

[Figures 1 and 2 from Kolstad et al. 2005] in Section 2.5.1.)

In several of the case-control studies, reliance is placed on self-administered or in-person

questions to establish either jobs held or potential exposures among living respondents

combined with assignation of exposure by a member of the research team (Dumas et al.

2000, Flodin et al. 1986, Gérin et al. 1998, Parent et al. 2000, Scélo et al. 2004, Seidler et


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al. 2007), which raises questions about the accuracy of recall by respondents (as well as

the possibility of misclassification bias in studies where disease status was known to

researchers, as discussed below). In the breast cancer mortality case-control study by

Cantor et al. (1995) potential exposure was assigned based only on usual occupation

listed on the death certificate, and in the ecological study by Coyle et al. (2005) an

indirect measure of exposure based on toxic releases in the county of residence was used;

this method of estimating individual exposure is considerably less precise than the use of

usual job titles in the case-control studies. In both studies, the likelihood of

misclassification of cumulative styrene exposure is particularly high.

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Classification of workers by individual job titles (McMichael et al. 1976a, Ruder et al.

2004) or job-exposure matrices (Bond et al. 1992, Delzell et al. 2001, Kogevinas et al.

1994a, Matanoski et al. 1997, Santos-Burgoa et al. 1992, Seidler et al. 2007, Wong et al.

1994) may, at least partly, have reduced misclassification of exposure. [Exposure

classification by job-exposure matrices is preferable since workers may experience

different exposures within given departments according to the particular job peformed

and may move between one job and another within and across departments.] However, in

a validation test within the styrene-butadiene rubber industry, styrene exposure ranks

correlated poorly with styrene measurements (Matanoski et al. 1993), clearly illustrating

that it may be difficult to obtain valid exposure estimates for styrene in this industry. If

exposure ranks and actual measurements correlate poorly, this would tend to attenuate

any apparent risk and bias the findings towards the null. Macaluso et al. (2004) generally

found estimates of styrene exposure in the styrene-butadiene rubber industry to be lower

than industrial hygiene measurements but did not conduct a thorough validation of their

exposure estimates.

In the Danish studies of the reinforced-plastics industry, duration of employment was

abstracted from national pension fund records. Based on a small validation study, the

estimates of duration of employment from the national pension fund records did not

correlate well with information obtained from a questionnaire from a sub-sample of 671

employees from 8 companies. It was determined that up to 40% of the workers classified

as short-term workers by the national pension fund were classified as long-term workers


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by the questionnaire, while the opposite misclassification occurred among 13% of the

workers classified as long-term by the national pension fund (Kolstad et al. 1994).

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The study by (Anttila et al. 1998) was the only one that relied on individual

measurements of exposure; exposure status thus was well documented for these subjects.

On the other hand, other studies have shown considerable intra-individual (Symanski et

al. 2001) and intra-company (Kolstad et al. 2005) variability in styrene exposure in the

reinforced-plastics industry; group-level exposure assessment (Delzell et al. 2001,

Kogevinas et al. 1994a, Macaluso et al. 1996, Matanoski et al. 1997) may therefore be

preferable (Armstrong 1998).

Such misclassification of styrene exposure was independent of health outcome and thus

would be expected to be nondifferential and to bias any measures of association towards

no effect. Exceptions were (1) the studies reported by Delzell and colleagues using the

revised exposure assessment (Delzell et al. 2001, Graff et al. 2005, Delzell et al. 2006),

because the investigators were aware of the employment histories of the workers who had

died of leukemia when they revised their exposure estimates, and (2) the population or

clinic-based, case-control studies, because they relied on patients’ retrospective

descriptions of exposures or working conditions (Dumas et al. 2000, Flodin et al. 1986,

Gérin et al. 1998, Parent et al. 2000, Scélo et al. 2004, Seidler et al. 2007). Kolstad et al.

(1994) compared exposure data obtained from employers in the reinforced-plastics

industry with those obtained from dealers in raw materials and found indications that

employers’ reports were not independent of health outcome for some companies; the

employers’ reports therefore were omitted from the analyses.

3.6.3 Other possible biases and confounding 23

As noted above, the potential exists for coexposure to other chemicals in the various

styrene-based industries. Apart from limitations in evaluating exposure to such chemicals

addressed above, there are limitations in the ability of the statistical modeling methods

used to adjust for such exposures to disentangle the effects of individual chemicals,

particularly in cases where multiple co-exposures occur such as in the styrene monomer

and polymer industry, where interaction effects might occur, and/or where a high degree


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of correlation between exposures is observed, such as in the styrene-butadiene industry.

Several studies adjusted for butadiene (Delzell et al. 2001, Delzell et al. 2006, Graff et al.

2005, Santos-Burgoa et al. 1992) and DMDTC (Delzell et al. 2001, Delzell et al. 2006,

Graff et al. 2005). Graff et al. (2005) noted that styrene, butadiene, and DMDTC

exposure are highly correlated, and it is difficult to separate the effects of one agent from

the other two agents. Butadiene is classified as a known human carcinogen by IARC and

the NTP, and is considered to be a risk factor for leukemia (IARC 1999, NTP 2004).

DMDTC is less strongly correlated with styrene exposure than butadiene, 0.6 compared

with 0.8 (Delzell et al. 2001). It is considered to be an immune system depressant (T-cell)

(Delzell et al. 2006), but its carcinogenicity has not been evaluated outside the studies in

the styrene-butadiene industry. Although there is potential exposure to benzene in the

styrene-butadiene rubber industry, it was not considered to have an impact on leukemia

based on studies by Macaluso et al. 1996 (Delzell et al. 2006).

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In the styrene monomer and polymer industry, the confounding effects due to multiple

co-exposures are difficult to distinguish from the potential effect of styrene (particularly

as some co-exposures, such as benzene and ethylbenzene, are known or suspected

carcinogens). In the case-control studies, there was generally little or no attempt to adjust

ORs by other exposures or to take into account multiple comparisons due to the large

number of potential exposures investigated.

Analyses in the studies reviewed generally were adjusted for age, sex, and, in some

studies, calendar year. In addition, some studies included information about years since

hiring (Delzell et al. 2001, Delzell et al. 2006, Graff et al. 2005) duration of employment

(Matanoski et al. 1997, Wong et al. 1994), and race (Matanoski et al. 1997). Very few

studies analyzed data by year of first hire, which may be important because there is

evidence that overall exposures in the three industry sectors have been reduced over the

past several decades. Estimates of cumulative exposure that are based on recent

measurements or estimates of current exposure may not accurately reflect the higher

exposures experienced by older workers. Wong et al. controlled for smoking in their

nested case-control study of lung cancer in the reinforced-plastics industry, but this

apparently did not affect the relative risk estimates related to styrene exposure (Wong


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1990). Certain lifestyle factors and/or other occupational factors were included in

analyses of the clinic- and population-based, case-control studies (Dumas et al. 2000,

Flodin et al. 1986, Gérin et al. 1998, Parent et al. 2000, Scélo et al. 2004, Seidler et al.

2007), but not in the cohort-based studies. Internal analyses within the worker

populations are expected to be less sensitive to confounding by lifestyle factors, because

the populations are expected to be relatively homogenous with respect to socioeconomic

factors (Delzell et al. 2001, Kogevinas et al. 1994a, Kolstad et al. 1995, Kolstad et al.

1994, Wong et al. 1994). However, confounding cannot be ruled out, because little is

known about the causes of the majority of the malignant diseases studied.

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Short-term workers in the reinforced-plastics industry showed generally higher cancer

risk than long-term workers (Kogevinas et al. 1994a, 1995, Kolstad et al. 1994, Ruder et

al. 2004, Wong et al. 1994). This might be because short-term workers are mainly

assigned to jobs with high styrene exposure; however, no data are available to assess this

hypothesis. The finding might also be explained by the healthy-worker effect — that is, a

selection process by which workers who become unfit during employment tend to leave.

However, the healthy-worker effect is generally less for malignant diseases than for

chronic nonmalignant diseases (Arrighi and Hertz-Picciotto 1994). A third explanation

might be confounding because of differences in other risk factors between short- and

long-term workers. A separate study supported this hypothesis; it showed that Danish

short-term reinforced-plastics workers had been hospitalized for lifestyle-related health

conditions before employment in the industry more often than long-term workers

(Kolstad and Olsen 1999). Thus, confounding by factors related to lifestyle is a likely

explanation, at least to some extent, of the unexpected decline in risk with length of

employment. One way of handling such confounding would be by comparison with a

non-styrene–exposed group of short-term workers with expected comparable

socioeconomic status and lifestyle factors. Such analysis of the Danish reinforced-plastics

industry workers showed a statistically nonsignificant increased risk of leukemia (RR =

1.89, 95% CI = 0.78 to 4.59) (Kolstad et al. 1994).


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3.7 Summary of previous evaluations (IARC and Cohen et al.) 1

As mentioned in the introduction, the 1979 and the 1994 IARC working groups

characterized the evidence available to them at the time on carcinogenicity of styrene in

humans as “inadequate” (IARC 1979, 1994a). The 2002 working group upgraded the

human evidence to “limited” (IARC 2002).

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In its 2002 evaluation of the human data, IARC considered case reports; cohort studies of

workers in the reinforced-plastics, styrene-butadiene rubber, and the styrene monomer

and polymer industries; nested case-control studies within the styrene-butadiene rubber

industry; biomonitoring of workers for styrene exposure; environmental exposure of

students to styrene; and clinic- and population-based, case-control studies of acute

myeloblastic leukemia and 15 major cancer sites.

IARC (2002) regarded data from the reinforced-plastics industry as the most informative,

because workers in that industry were exposed to the highest levels of styrene and had

less potential for exposure to other substances within the occupational setting than the

other cohorts studied. The IARC evaluation emphasized a small, nonsignificantly

increased incidence of leukemia among Danish reinforced-plastics workers and a

statistically significant excess among those workers with the earliest first years of

employment, the highest styrene exposure levels, or latency of at least 10 years.

However, among all workers exposed for 1 year or more, the incidence of leukemia was

not increased. In a European multinational cohort of reinforced-plastics workers (that

partially overlapped with the Danish study), mortality from lymphatic and hematopoietic

neoplasms was not increased, based on comparison with national reference rates.

However, in an internal analysis using the unexposed workers as the comparison group,

mortality was increased in exposed workers after 20 years since the first exposure to

styrene and also increased with increasing intensity of exposure, but not with increasing

cumulative exposure to styrene. A large U.S. mortality study of reinforced-plastics

workers found no overall excess of lymphohematopoietic malignancies. IARC stated that

problems with mortality ascertainment in the European study and underestimation of

duration of exposure in the Danish study might have influenced the findings.


9/29/08 165

Studies of the styrene monomer and polymer industry showed weak association between

styrene exposure and lymphohematopoietic cancers, and studies of the styrene-butadiene

rubber industry showed increasing mortality from leukemia with increasing cumulative

exposure to styrene. IARC considered these findings difficult to interpret because of

potentially confounding coexposures; in the styrene-butadiene rubber industry, styrene

exposure was highly correlated with butadiene exposure. IARC mentioned increased

risks of rectal, pancreatic, and nervous system cancers in some studies, but considered

those findings of limited importance.

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Cohen et al. (2002) reviewed epidemiologic studies relevant to the carcinogenicity of

styrene. The authors concluded that the balance of epidemiologic evidence did not

suggest a hazard of cancer in humans from exposure to styrene. The authors emphasized

that there were no consistent patterns of increased risks for the various lymphatic and

hematopoietic cancers (NHL, Hodgkin’s disease, multiple myeloma, and leukemia)

across studies of the reinforced-plastics industry, which they considered the most

informative because subjects had high styrene exposure levels and few other potentially

confounding occupational exposures. They stressed the absence of exposure-response

patterns for these cancers. The only study identified as showing a statistically significant

increased risk of lung cancer was that of Wong et al. (1994), and that risk was confined

to short-term workers, indicating confounding related to socioeconomic status. Cohen et

al. also stressed the finding of no increased risk of lung cancer in the European study

conducted by Kogevinas et al. (1994a). As general problems of the studies reviewed, the

authors emphasized nondifferential misclassification of exposure with respect to disease

outcome and imprecise diagnoses in studies relying on death certificates. Cohen et al.

mentioned that some other cancers (of the esophagus, pancreas, urinary tract, and genital

organs) showed increased risks in some studies, but they did not consider them related to

styrene exposure, because the increases were small and statistically nonsignificant, and

they did not concentrate in groups with high exposure.

3.8 Summary of the findings for selected cancer sites 28

The results for 12 separate study populations are presented in Table 3-8 for major cancer

sites. This tabulation did not include the study by Coggon et al. (1987) because this


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population was included in the Kogevinas et al. (1994a) study of the European

reinforced-plastics industry. It includes the Kolstad studies (one report on

lymphohematopoietic cancers from 1994 and one report on solid cancers from 1995), but

it should be emphasized that 15,867 of the 36,610 Danish workers were also included in

the Kogevinas et al. study (1994a). The findings by Ruder et al. (2004) are also included,

but not those of Okun et al. (1985), similarly, those of Wong et al. (1994) but not Wong

et al. (1990) are included, and Bond et al. (1992) but not Ott et al. (1980) are described

because the reports included were based on the last and longest follow-up of the same

cohorts.

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For the styrene-butadiene rubber industry, the results for the cohort described in

Sathiakumar et al. 2005 and Delzell et al. 2006 are included, but results from other

studies of this industry were omitted due to major overlap of study populations

(Matanoski et al. 1997, Matanoski et al. 1993, Matanoski et al. 1990, Matanoski and

Schwartz 1987, Meinhardt et al. 1982, Santos-Burgoa et al. 1992), or because of shorter

follow-up (Sathiakumar et al. 1998), or because they did not tabulate results for the major

cancer sites but focused on exposure response for leukemia and other

lymphohematopoietic cancers (Delzell et al. 2001, Delzell et al. 1996, Graff et al. 2005,

Macaluso et al. 1996).

[This tabulation suggests that the strongest indications of consistently increased risks

across the individual studies were for cancer of esophagus, pancreas, larynx, lung, and

lymphohematopoietic tissues (NHL, Hodgkin’s disease, multiple myeloma, and

leukemia)]. Pooled results for these selected cancers obtained from studies of workers in

the reinforced-plastics industry are presented in Table 3-9. For each study, results for the

well-defined worker category with the highest styrene exposure (defined by title or task)

are presented when possible. Table 3-10 presents these results for workers in the

reinforced-plastics industry.

Among all 85,000 workers included in studies of the reinforced-plastics industry, 2,238

cases of cancer were identified from death certificates and cancer incidence registrations,

which is close to the expected number (2,210.5). In this tabulation (Tables 3-9 and 3-10)


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the male Danish workers (results were not presented for female workers [(Kolstad et al.

1995, Kolstad et al. 1994)] were omitted from the dataset of the European reinforced-

plastics industry (Kogevinas et al. 1994a). This was done based on country-specific

findings published in an IARC report describing details of the European study

(Kogevinas et al. 1994b). [This was done to eliminate overlap between the Danish and

the European datasets and thus erroneously pooled estimates. Another option would be to

exclude the total Danish dataset (as Cohen et al. 2002 did in their review), but this would

mean that the results based on that part of the Danish population not included in the

European data set would be left out. Furthermore, the Danish study reported incidence

data while the European study only reported mortality data, and incidence data is

regarded the most relevant for several of the cancers studied with relatively low

mortality. Therefore, inclusion of the Danish population and exclusion of the male

Danish workers from the European study when pooling observed and expected number of

cases, is expected to have improved the validity of the overall evaluation.]

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[The data in Tables 3-8 to 3-10 permit a general comparison of multiple studies and the

identification of trends in the data (e.g., several studies reporting nonsignificant increases

in a specific site). However, there are several limitations inherent in pooling data across

different studies. Study populations may differ with respect to, for example, the size and

type of population studied, their racial or age composition, inclusion and exclusion

criteria (such as minimum duration of employment or exposure time), latency periods,

duration of follow-up, the nature and intensity of exposure, and the type of cases reported

(incidence vs. mortality). In addition, studies clearly vary in quality, e.g., with respect to

the power of the study, (especially for specific cancer sites) and which, if any, potentially

confounding variables (e.g., potential exposure to other carcinogens) are adjusted for.

This methodology also does not incorporate information on subgroup analyses (such as

exposure-response relations) that may be important in evaluating causality. However, as

mentioned previously, one of the major limitations of the body of literature is the small

numbers of highly exposed workers, which limits the ability to detect an effect, especially

for uncommon tumors. This approach (summing observed and expected cases of highly

exposed workers from all studies) facilitates the evaluation of the relationship between

styrene exposure and cancer risk of these tumors.]


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Table 3-8. Relative occurrence of cancer in 12 cohort studies of populations exposed to styrene (total study populations)

Reinforced-plastics industry

Styrene monitored workers SBR industry Styrene monomer & polymer industry

Environ. exp.


Loughlin et al. 1999

Cancer site

Ruder et al. 2004

Wong et al. 1994

Kolstadet al. 1994, 1995

Koge-vinaset al. 1994a

Anttila et al. 1998

McMi-chael et al. 1976

Sathia-kumaret al. 2005c

Delzell et al. 2006

Frentzel-Beyme et al. 1978

Bond et al. 1992

Nicholson et al. 1978 SMR SIR M F

all cancer + + – (–) – – (–) (–) (–) (+) – buccal cavity & pharynx

(–) (–) – – –

lip (+) tongue (–) salivary gland

(+)

mouth (+) pharynx (–)

digestive sys.

(+) (–) (+) (–)

esophagusd + + (–) (–) (–) (–) (+) stomach (+) (–) (–) (–) (+) + (–) (–) (+) small intestine

(+)

large intestine

(+) (–) (–) (–) (-) (–) (–)


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Environ. exp.



Cancer site

Ruder et al. 2004

Wong et al. 1994



Anttila et al. 1998



Delzell et al. 2006


Bond et al. 1992


intestine except rectum

(–)

rectum (+) (–) – + (–) (–) large intestine & rectum

(–) (+)

liver & gallbladder

(–) (+) (–) (–)

liver (–) (+) (–) gallbladder (–) pancreasd (+) (+) (+) (+/–) (+) (–) (+) (–) peritoneum (+)

respiratory sys.

(+) + (–) (–)

nose & nasal cavities

(+) (–)

larynxd (+) (+) (+) (–) (–) + lungd (+) + (+) (–) (–) – (–) (–) (–) (+) (+) (–)


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Environ. exp.



Cancer site

Ruder et al. 2004

Wong et al. 1994



Anttila et al. 1998



Delzell et al. 2006


Bond et al. 1992


pleura (+) mediastinum (+) breast (–) (–) – (–) (–) female genital organs

(+) +e (+)

uterus (+)e cervix + (–) ovary (+) (+)

male genital organs

prostate + (+) (–) (+) (–) (+) (–) testis (–) (+) (–) (–) external male genital organs

(+)

urinary organs

(+) (+)

kidney (+) (+) (–) (–) (–) (–) (–)


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Environ. exp.



Cancer site

Ruder et al. 2004

Wong et al. 1994



Anttila et al. 1998



Delzell et al. 2006


Bond et al. 1992


bladder (+) (–) (+) (–) (–) (–) (–) skin (–) (–) melanoma (–) other skin (–)

eye – (–) brain & nervous sys.

(+) (–) (–) – (+) (–) (–)

thyroid (+) (–) (–) other endocrine glands

(+)

bone (–) (+) connective tissue

(–) (–)

all LHd (–) (–) (+) (–) (–) + (+) (+) (+) (+)f (–) all lymphomad

(–) + +

NHLd (–) (–) (+) (–) (+/–) (+) (+) (+) Hodgkin’s diseased

(–) (–) (+) (–) (+) (+) (+) (–) (+) (–)


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Environ. exp.



Cancer site

Ruder et al. 2004

Wong et al. 1994



Anttila et al. 1998



Delzell et al. 2006


Bond et al. 1992


multiple myelomad

(–) (–) (–) (+) (–)

leukemiad (–) (–) (+) (+) +c (+) (+) (–) (–) (+) (+) (–) + = Statistically significant excess of cancer; (+) = statistically nonsignificant excess of cancer; – = statistically significant deficit of cancer; (–) = statistically nonsignificant deficit of cancer; (+/–) = no excess/deficit of cancer, i.e., SMR = 1.0. a Male Danish workers were excluded from the European data set in this calculation to eliminate overlap between the Danish and the European datasets. bResults based on exposure ratios (colorectal, prostate, bladder, respiratory cancers) or risk ratios (stomach, all lymphohematopoietic, leukemia). cData are from the follow-up reported by Sathiakumar et al. 2005 of 17,924 workers from 1944 to 1998 except for esophagus, which was not reported in the 2005 analysis, so is from the earlier follow-up of the smaller cohort (N = 15,649) followed up to 1991 and reported in Sathiakumar et al. 1998. dCancer site selected for more thorough evaluation, see Tables 3-8 and 3-9. eThe paper reported statistically significant increases for cervix (SMR = 2.835, 1.359–5.213, P < 0.01) and other female genital organs (SMR = 2.016, 1.074–3.448, P < 0.05), while uterus (SMR = 1.973, 0.985–3.531) approached statistical significance. fIncident cases overlap with deaths; 2 cases of lymphohematopoietic cancer (one lymphoma and one leukemia) were not recorded as the underlying cause of death.


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3.8.1 Esophageal cancer 1

Reinforced plastic workers: Statistically significantly increased risks were observed in the

two U.S. studies (Ruder et al. 2004 and Wong et al. 1994), and a nonsignificantly

increased risk was observed for laminators, but not for workers with unspecified tasks or

other exposed jobs, in the European cohort (Kogevinas et al. 1994a). The SIR in the

Danish study was close to unity (Kolstad et al. 1995). Among the worker categories with

the highest potential styrene exposure ― laminators (Kogevinas et al. 1994a, Ruder et al.

2004), open-process mold workers (Wong et al. 1994), and workers at companies

employing 50% to 100% laminators (Kolstad et al. 1995) – a total of 14 cases of

esophageal cancer were observed (≥ 7.2 expected

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4; see Table 3-10). A statistically

nonsignificant trend toward increased esophageal cancer mortality with increasing

cumulative styrene exposure was seen among European reinforced-plastics workers

(Kogevinas et al. 1994a), and mortality was highest at ≥ 20 years since first exposure.

Mortality was increased (SMR = 2.74, 95% CI = 0.004 to 22.3, 1 observed death) among

workers from Washington state with high exposure for more than 1 year (Ruder et al.

2004).

Other industries: Among workers in the styrene-butadiene rubber industry, the SMR for

esophageal cancer was close to unity (SMR = 0.94, 95% CI = 0.68 to 1.26, 44 observed

deaths) (Sathiakumar et al. 1998). Among styrene monomer production workers, 4 cases

were identified, compared with 5.1 expected (Bond et al. 1992, Hodgson and Jones

1985). [Evaluation of site-specific cancer risks in this industry is limited by the small

numbers of subjects; three of the four studies had low numbers (fewer than 20) of

expected and observed cases of all malignant tumors.] Risk estimates for esophageal

cancer were not reported for the biomonitoring study (Anttila et al. 1998) or the study of

environmental exposure to styrene-butadiene (Loughlin et al. 1999).

With respect to the case-control studies, only the study of Gerin et al. (1998) investigated

esophageal cancer, in association with potential exposure to four solvents (benzene,

toluene, xylene, and styrene). No statistically significant association between esophageal

4 “≥” is used when at least one study reported the number of observed cases but not expected cases.


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cancer and styrene exposure was observed (OR adjusted for demographic, socioeconomic

and lifestyle factors = 1.4 [95% CI = 0.5 to 3.8, based on 5 cases] for “medium/high

exposure”).

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3.8.2 Pancreatic cancer 4

Reinforced-plastics workers: Increased risks (1 significant and 2-nonsignificant) of

pancreatic cancer were observed among the high-exposure groups in three of the four

reinforced-plastics worker populations (Kogevinas et al. 1994a, Kolstad et al. 1995,

Ruder et al. 2004), but not in the fourth (Wong et al. 1994) (see Table 3-8. A total of 34

cases were registered across all four populations (high-styrene–exposure groups)

compared with 19.2 expected (Table 3-10 [corresponding to an SMR value of 1.77 (95%

CI = 1.23 to 2.47)]. In internal analyses, Kolstad et al. (1995) reported significant risks of

pancreatic cancer among individuals with probable high styrene exposure (workers from

plants employing 50% to 100% laminators), and among individuals exposed to styrene

for greater than one year. The risk of pancreatic cancer increased with increasing

cumulative styrene exposure (P = 0.068) (Kogevinas et al. 1994a), and a slightly higher

risk was seen among long-term than among short-term workers and earlier start dates

(Kolstad et al. 1995), but not in all studies (Ruder et al. 2004).

Other industries: In styrene-butadiene rubber industry workers, the SMR was 0.87 (95%

CI = 0.68 to 1.08; 76 observed deaths) (Sathiakumar et al. 1998). The findings from the

styrene monomer and polymer industry were divergent; decreased mortality (non-

significant) was reported by Bond et al. (1992) and an increased mortality (non-

significant) was reported by Frentzel-Beyme et al. (1978) and the pooled number of cases

was less than expected (7 observed vs. 11 expected). The biomonitored workers (Anttila

et al. 1998) showed a 3-fold increased risk of pancreatic cancer (SIR = 3.64; 95% CI =

0.75 to 10.6, 3 cases) 10 years or more after the first measurement. No risk estimate was

reported in the environmental exposure study.

No increased risk of pancreatic cancer (based on 1 exposed case and 22 exposed controls)

was reported in the population-based, case-control study reported by Gérin et al. (1998).


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3.8.3 Laryngeal cancer 1

Reinforced-plastics workers: Among all reinforced-plastics workers, 36 cases of

laryngeal cancer were observed (vs. 32.7 expected) (Table 3-9), yet only 3 cases were

identified among the workers classified with the highest styrene exposure (vs. ≥ 1.9

expected) (Table 3-10).

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Other industries: The SMR value was non-significantly decreased (SMR = 0.71, 95% CI

= 0.41 to 1.13, 17 observed deaths) in the styrene-butadiene rubber industry (Sathiakumar

et al. 2005). In the styrene monomer and polymer industry, only 1 case (death) was

reported (vs. 2.9 expected) (Bond et al. 1992). Hodgson and Jones (1985) reported an

excess of incidence cases (3 observed vs. 0.5 expected, P = 0.041); however, no mortality

was reported. The authors stated that laryngeal cancer is often amenable to treatment.

Risk estimates were not calculated in the biomonitoring or environmental studies, and

there were no case-control studies evaluating laryngeal cancer.

3.8.4 Lung cancer 14

Reinforced-plastics workers: Lung cancer risk was significantly increased among U.S.

workers (Wong et al. 1994), and increased but not statistically significant among workers

from Denmark (Kolstad et al. 1995) and Washington state (Ruder et al. 2004). Among

the highest-styrene–exposure group in the reinforced-plastics industry, 158 cases of lung

cancer were observed, compared with 151.5 expected (Table 3-10). Lung cancer risk was

lower among styrene-exposed workers in a nested case-control study that controlled for

smoking, in long-term workers, and among workers with higher cumulative styrene

exposure (Kogevinas et al. 1994a, Kolstad et al. 1995, Ruder et al. 2004, Wong et al.

1994).

Other industries: In the styrene-butadiene rubber and the styrene monomer and polymer

industries, fewer cases were observed than expected. No increased risk of lung cancer

was seen in workers biomonitored for styrene exposure (Anttila et al. 1998), in the

styrene-butadiene rubber industry (McMichael et al. 1976a, Sathiakumar et al. 1998), or

in the styrene polymer manufacturing industry (Frentzel-Beyme et al. 1978) (see Table 3-

8). No significant association with lung cancer was observed among potentially styrene-


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exposed cases in a population-based, case-control study by Scelo et al. (2004) or the

population-based study of Gerin et al. (1998), although the power to detect an effect in

the latter study is low.

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3.8.5 Lymphohematopoietic cancers 4

Not all studies reporting on lymphohematopoietic cancers analyzed by type. In addition,

the power to detect increased risks for subtypes of lymphohematopoietic cancers is

limited by the small number of total lymphohematopoietic cancers observed in some

cohorts.

3.8.5.1 All lymphohematopoietic cancers combined 9 Reinforced-plastics workers: Kolstad et al. 1994 reported a non-significantly increased

incidence for all lymphohematopoietic malignancies (SIR = 1.20; 95% CI = 0.98 to 1.44,

112 observed cases) among Danish workers (which overlaps with the international study

reported by Kogevinas et al. (1994a, 1993). No increase in lymphohematopoietic cancer

mortality was observed for the two U.S. studies (Ruder et al. 2004, and Wong et al.

1994). Among all workers in the reinforced plastic industry, 196 cases were observed

compared with 199.2 expected (Table 3-9). Observed among the high-styrene–exposure

groups in the reinforced-plastics industry were 52 cases of any lymphohematopoietic

malignancy (53 expected) (see Table 3-10). In the largest study (the multi-country) the

risk of all lymphohematopoietic malignancies increased with average exposure (P =

0.019) and time since first exposure (P = 0.012), but did not increase with increasing

cumulative styrene exposure (Kogevinas et al. 1994a). No increased risk was observed

with duration of employment in the other studies (Kolstad et al. 1994, Ruder et al. 2004,

Wong et al. 1994).

Styrene-butadiene rubber workers: The principal methodological challenge in these

studies lies in teasing out possible independent or synergistic effects of butadiene, which

is highly correlated with styrene exposure in this industry. 1,3-Butadiene is listed as

known to be a human carcinogen in the 11th Report on Carcinogens (NTP 2004). In the

synthetic rubber industry, McMichael et al. (1976a) reported a significant increase in the

age-standardized relative risk of all lymphohematopoietic cancers (RR = 6.2, 99% CI =


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4.1 to 12.5) among workers engaged in synthetic rubber tire manufacture (primarily

styrene-butadiene), [but no adjustment for other exposures was attempted].

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In the cohort of styrene-butadiene rubber workers established by Delzell and colleagues,

a slightly increased mortality from all lymphohematopoietic malignancies (SMR = 1.06,

95% CI = 0.90 to 1.23, 162 observed deaths) was observed in the 1998 follow-up by

(Sathiakumar et al. 2005). This cohort included styrene-butadiene rubber workers from a

smaller cohort reported by Meinhardt et al. and most of the workers from a larger cohort

studied by Matanoski and coworkers. There were numerous publications on both cohorts

or subpopulations of the cohorts (Matanoski and Delzell), and interpretation of the

studies are complicated by overlapping populations, different exposure assessments and

different types of analyses.

Risk estimates for quantitative exposure to styrene and the risk of lymphohematopoietic

cancers (combined) was not reported in the most recent updates of the most

comprehensive cohort (e.g., Delzell et al. 2006); however, it was studied in two nested

case-control studies from the Matanoski cohort, which reported findings for workers

employed from 1943 to 1976 and followed until 1982. The nested case-control study (59

cases and matched controls) reported by Santos-Burgoa et al. (1992) found non-

significant increases for cumulative exposure to styrene (greater than average exposure)

and lymphohematopoietic mortality using matched and unmatched analyses; however,

the magnitude of the OR was decreased in matched models that controlled for butadiene

exposure. The second case-control study (58 cases and 1,242 controls) found a two-fold

significantly increased risk for lymphohematopoietic cancers (combined) and time-

weighted average (working lifetime) exposure to 1-ppm styrene after taking into account

butadiene exposure and other variables in a step-down logistical regression analysis

(Matanoski et al. 1997). This analysis used an exposure assessment based on

measurements of styrene air levels (taken in 1978 to 1983) and used controls sampled

without individual matching by plant and other variables, whereas exposure was assessed

by job-exposure matrix in the study by Santos-Burgoa et al.


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Other industries: In the largest study of the styrene monomer and polymer industry, the

risk of all lymphohematopoietic malignancies increased with increasing duration of

exposure (if workers with < 1 year are compared with those with > 1 year of exposure)

but not with increasing styrene exposure level (Bond et al. 1992). Among all workers at

the four styrene monomer and polymer plants studied, there were 34 deaths due to

lymphohematopoietic malignancies, compared with 23.1 expected (Bond et al. 1992,

Frentzel-Beyme et al. 1978, Hodgson and Jones 1985, Nicholson et al. 1978). Among

workers biomonitored for styrene exposure, the incidence of all lymphohematopoietic

malignancies was not increased (SIR = 0.39, 95% CI = 0.05 to 1.40, 2 cases) (Anttila et

al. 1998). No cases of lymphohematopoietic cancer occurred 10 years or more after the

first measurement, but the study included only 2 cases. In the study of environmental

exposure to styrene (Loughlin et al. 1999), a non-significantly increased risk of all

lymphohematopoietic malignancies was reported among men.

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3.8.5.2 Leukemias 14 Reinforced plastic workers: Among the reinforced-plastics workers, Kolstad et al. 1994

reported a non-significant increased incidence in leukemia (SIR = 1.22, 95% CI = 0.88 to

1.65, 42 observed cases) among Danish workers (which overlaps with the international

study reported by Kogevinas et al. (1994a, 1993). Kogevinas et al. reported non-

significant increases for myeloid leukemia mortality, but no increase with increasing

average or cumulative exposure was observed; a non-significant trend was observed with

time since first exposure (P = 0.094). In the Danish study, significantly increased

mortality from leukemia was observed among workers with more than 10 years after first

styrene exposure and for workers with earlier years of first employment (Kolstad et al.

1994). No relationship between cumulative exposure or duration was observed among the

U.S. workers reported by Wong et al. (1994). Among the high-styrene–exposure groups

in the reinforced-plastics industry, a total of 19 cases of leukemia was observed (19.6

expected) (see Table 3-10). In analyses of subtypes of leukemia, the risk of myelogenous

leukemia (chronic and acute) was slightly higher than for all leukemia (Kogevinas et al.

1994a), and increased risk was also seen for myeloid leukemia with chromosomal

aberrations in a nested case-control study of the Danish workers (Kolstad et al. 1996).


9/29/08 179

Styrene-butadiene rubber workers: McMichael et al. (1976a) reported a statistically

significant increase in the age-standardized risk for lymphatic leukemia (RR = 3.9, 99%

CI = 2.6 to 8.0) among rubber tire workers engaged in synthetic rubber manufacture

(primarily styrene-butadiene) [but no adjustment for other exposures was attempted].

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In the latest follow-up (to 1998) of the most comprehensive cohort of styrene-butadiene

workers, the SMR for leukemia among all workers was 1.16 (95% CI = 0.91 to 1.47, 71

deaths) (Sathiakumar et al. 2005). Compared with workers with other combinations of

duration of employment and time since hire, the highest risk of leukemia in this cohort

was observed among workers with > 10 years of employment and 20 to 29 years since

hire (SMR = 2.58, 95% CI = 1.56 to 4.03, 19 deaths). Among this subgroup, those who

had been hired between 1950 and 1959 had the highest risk of leukemia (SMR = 3.92,

95% CI = 1.96 to 7.03, 11 deaths (Delzell et al. 2006). Statistically significant increased

risks of leukemia (SMR ranging from 2.58 to 4.31) were observed among workers

involved in production (polymerization and coagulation) job groups) and labor

(maintenance and laboratories job groups) (Sathiakumar et al. 2005). (Note that

production and maintenance workers had high exposure to both styrene and butadiene,

and coagulation workers had low to moderate exposure to styrene, but only background

exposure to butadiene.) Significant SMRs (approximately 2-fold increased) were also

reported among the two highest categories of cumulative levels of styrene exposure.

Exposure to styrene and leukemia risk were evaluated in the two nested case-control

studies from the Matanoski cohort and in several reports from the Delzell cohort [note

that these cohorts overlap]. Santos-Burgoa et al. (1992) reported a significantly increased

risk of leukemia for cumulative exposure greater than average exposure in both matched

and unmatched analysis; however, the risk was no longer significant after controlling for

butadiene exposure. In the nested case-control study from the Matanoski cohort, no

significant risks were found for leukemia and 1-ppm time-weighted average exposure to

styrene; however, a significant association between leukemia and cumulative exposure

was found in a final model that included styrene, butatdiene exposure, and duration of

employment (Matanoski et al. 1997).


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Graff et al. (2005) and Delzell et al. (2006) conducted a series of internal analyses in

which exposure to butadiene and DMDTC were adjusted for either in models of

cumulative exposure or in analyses of cross-classified categories of styrene and butadiene

exposure, for the 1998 update of the cohort established by Delzell. Statistically

nonsignificant increases in the relative risk of leukemia for categories of styrene and

butadiene exposure or quartiles of cumulative exposure to styrene in the single-, and two-

chemical models; however, the RRs were below one in the three-chemical model. [There

was a trend towards higher risk with increasing exposure to styrene alone and when

adjusted for butadiene and styrene; however, tests for trend, were not reported by the

authors.] In a similar analysis using cumulative exposure due to styrene total peaks > 50

ppm and butadiene total peaks > 100 ppm, increasing risks of leukemia with increasing

levels of exposure were observed in single-, two- and three-chemical models. In external

analysis of cumulative exposure to styrene in this cohort, significantly increased SMRs

were observed for the two highest categories of styrene exposure; [however, there was no

adjustment for exposure to butadiene or DMDTC.]

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Statistically nonsignificant increases in relative risk were observed in internal analyses by

subtypes of leukemia (CLL, AML, and other) for terciles of styrene exposure; no

increasing risks with exposure were observed for CML (Graff et al. 2005, Delzell et al.

2006). (These analyses were restricted to workers over 40 years of age and, in some

cases, to > 20 years since hire). In external analyses of CLL, AML, and CML, non-

significant increases in CML and CLL were observed, and CLL was significantly

increased at cumulative styrene exposures exceeding 61.1 ppm-years (SMR = 3.10, 95%

CI = 1.01 to 7.24, 5 deaths).

Other studies: Among all workers at the four styrene monomer and polymer plants

studied, slightly increased mortality was seen for leukemia (10 observed deaths vs. 8.7

expected (Bond et al. 1992, Frentzel-Beyme et al. 1978, Hodgson and Jones 1985,

Nicholson et al. 1978). A statistically nonsignificant increase in leukemia was observed

in the U.S. cohort of styrene monomer and polymer workers (SMR = 1.18, 95% CI =

0.54 to 2.24, 9 deaths) (Bond et al. 1992). In the study of environmental exposure to


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styrene (Loughlin et al. 1999), non-significant increases in leukemia were reported

among men.

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The 2 case-control studies that addressed leukemia (Flodin et al. 1986, Guenel et al.

2002) had small numbers of exposed subjects (Guenel – 2 cases of leukemia and 9

controls, and Flodin – 3 cases of myeloid leukemia and 1 control), [precluding any firm

conclusions (despite the high OR found in the study of Flodin et al.)].

3.8.5.3 Other lymphohematopoetic cancers 7 Reinforced plastic workers: Among reinforced-plastics workers, statistically

nonsignificant elevations in lymphomas were observed by Kolstad et al. (1994) (SIR =

1.33, 95% CI = 0.96 to 1.80, 42 cases) and Kogevinas et al. (1994a) (among laminators)

(SMR = 1.40, 95% CI = 0.56 to 2.88, 7 cases), but no increased risks were observed in

the smaller cohorts. Among the high-styrene–exposed groups in the entire industry, a

total of 14 cases of NHL (vs. 15.1 or more expected) and 11 cases of Hodgkin’s disease

(vs. 7.9 or more expected) were observed (Table 3-10). Kogevinas et al. reported that the

risk of malignant lymphoma increased with averge exposure (P = 0.052) and with time

since first exposure (P = 0.072), but not cumulative exposure.

Styrene-butadiene rubber workers: In the styrene-butadiene industry, several subtypes of

lymphohematopoietic cancers were investigated in the overlapping cohorts established by

Matanoski and Delzell et al. The nested case-control study from the Matanoski cohort of

58 lymphohematopoietic cases and 1,242 controls found two- to three-fold increased

risks for lymphoma, lymphosarcoma, and myeloma and styrene exposure (increase of 1

ppm in TWA) (Matanoski et al. 1997), and the risk of myeloma increased with increasing

cumulative exposure to styrene using the step-down regression analysis and taking into

account butadiene exposure and other variables. Styrene exposure was not associated

with Hodgkin’s disease. However, no associations between other types of

lymphohematopoietic cancers (lymphosarcoma, Hodgkin’s disease, and other lymphatic

cancers) were observed in the nested case-control study reported by Santos-Burgoa et al.

(1992).


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In the 1998 follow-up of the Delzell et al. cohort, no significant increases in risks were

observed for Hodgkin’s disease, multiple myeloma, or NHL (Delzell et al. 2006). No

significant increases in the risk of NHL, Hodgkin’s lymphoma, or multiple myeloma

were observed in subgroup analyses of years since hire and latency or by work group/job

area. Borderline significanty increased risks of NHL+CLL were observed among all ever-

hourly workers, and significantly increased risks were observed among these workers

with greater than 10 years of employment and 20 to 29 years or 30+ years since hire;

significantly increased risks were also observed for styrene-butadiene rubber workers in

polymerization and finishing workshops. Statistically significant SMRs for NHL or

NHL+CLL were also observed for the highest cumulative levels of styrene, but no

association was found between multiple myeloma and exposure to styrene. In internal

analyses, increasing risks of NHL and of NHL+CLL with increasing quartiles of styrene

exposure were observed before and after controlling for butadiene and/or DMDTC

exposure (although the trends were attenuated in models with DMDTC), but no one

quartile was significant (Graff et al. 2005, Delzell et al. 2006). In single-chemical

models, the risk for NHL and NHL+CLL also were increased at the two highest levels of

butadiene exposure; however, no increased risk was observed after controlling for

styrene, [suggesting that butadiene was not a risk factor for these cancers; however,

butadiene is a risk factor for leukemia. Tests for trend were not performed.] No such

trend was seen for multiple myeloma. Similar results were seen in SMR analyses. When

all lymphoid and all myeloid cancers were considered in two separate groups, no

significant increases in relative risks with increasing styrene exposure were observed in

single- or multiple-chemical models, with the exception of myeloid cancers at styrene

levels of 1.8 to < 61.1 ppm-years (RR = 2.6, 95% CI = 1.2 to 5.5, 13 deaths) (Delzell et

al. 2006).

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Other studies: Among all workers at the four styrene monomer and polymer plants

studied, slightly increased mortality was seen for lymphoma (11 observed deaths vs. 7.9

expected) and leukemia (10 observed deaths vs. 8.7 expected (Bond et al. 1992, Frentzel-

Beyme et al. 1978, Hodgson and Jones 1985, Nicholson et al. 1978). Among workers

biomonitored for styrene exposure, the incidence of Hodgkin’s disease was slightly, but


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nonsignificantly increased (SIR = 1.89, 95% CI = 0.23 to 6.84, 2 cases) (Anttila et al.

1998).

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The population and clinic-based, case-control studies evaluated different types of

lymphohematopoietic cancer and, as discussed above, were limited principally by

potential misclassification of exposure. The population-based, case-control study by

Seidler et al. (2007) included a sufficient number of cases and controls, but no significant

increase in malignant lymphoma was found and no trend with increasing exposure

detected. Non-significant increases in non-Hodgkin’s lymphoma (8 cases and 19

controls) and in Hodgkin’s lymphoma (2 cases and 19 controls) were observed in the

Canadian population-based, case-control study, but the number of exposed subjects was

too small to draw firm conclusions(Gérin et al. 1998).

3.8.6 Other sites 12

Findings are less consistent across cohort or case-control studies for other sites.

Significantly increased risks for cancer of the stomach (McMichael et al. 1976a), benign

neoplasms (which were brain tumors) (Loughlin et al. 1999), cervix and other female

genital organs (Wong et al. 1994) have been reported in a single study; however, other

studies reported either nonsignificantly increased or decreased risks. For other sites

(prostate, rectum, and urinary system), significant increases were reported in at least 2

studies or there was supporting exposure-response data.

Prostate: Ruder et al. (2004) reported a significant increase in prostate cancer mortality

among reinforced plastic workers; SMRs were elevated in both the high- and low-

exposure groups although the SMR was slightly higher in the high-exposure cohort.

Mixed results (nonsignificant increases or decreases) were observed in the other cohort

studies; however, Gerin et al. reported a significant risk (OR = 5.5, 95% CI = 1.4 to 21.8,

7 exposed cases and 3 controls) for medium to high styrene exposure in the Canadian

case-control study. In the most recent update of the Delzell et al. cohort, a slight but non-

significant increase in the SMR for prostate cancer was observed (SMR = 1.04, 95% CI =

0.88 to 1.21, 154 deaths) (Sathiakumar et al. 2005), but no increase in the relative risk of

prostate cancer was observed by increasing levels of cumulative styrene exposure in


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either single- or multiple-agent models (analysis restricted to workers 50+ years of age)

(Delzell et al. 2006).

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Rectal: A significantly increased incidence of rectal cancer was observed among the

biomonitored workers; the incidence was higher among individuals with > 10-year

follow-up, but did not increase with increasing lifetime urinary metabolite levels (Anttila

et al. 1998). A significant risk (OR = 3.9, 95% CI = 1.2 to 12.9, 5 exposed cases) was

also observed in the Canadian case-control study for substantial exposure to styrene

(Dumas et al. 2000). However, non-significant decreases or null results were reported in

most of the other studies. A slight but non-significant increase in the SMR for colorectal

cancer was observed by Sathiakumar et al. (2005) (SMR = 1.09, 95% CI = 0.94 to 1.25,

193 deaths). In an internal analysis of increasing exposure to styrene, the relative risk

exceeded 1.0 in 3 of 4 quartiles (1.2, 1.2, 0.6 and 1.5, respectively) but none of the

estimates were significant (Delzell et al. 2006).

Urinary: Ruder et al. reported an increase in urinary cancer mortality among the high-

exposure group of reinforced plastic workers from Washington state (SMR = 3.44, 95%

CI = 1.26 to 7.50, 6 observed deaths), and there was a trend towards increasing SMRs

with increasing duration of exposure in this group. In the multi-country European cohort

(Kogevinas et al. 1994a), the relative risk for kidney cancer increased with increased

cumulative exposure (although the test for trend was not significant), but decreased with

time since first exposure. The SMR was not elevated among the low-exposure group. An

increased risk of renal-cell cancer was also associated with exposure to styrene-butadiene

rubber in the population case-control study from Canada (Parent et al. 2000). Results

from other studies were not consistent, with some studies reporting nonsignificant

increases and others nonsignificant decreases.

Increased risk of breast cancer was suggested in an ecological study (Coyle et al. 2005),

which assessed styrene exposure by toxic release inventory data; [however, this study

was limited by the ecological design and poor characterization of styrene exposure,

which was based only on residence in counties with varying environmental toxic

releases]. A population-based, case-control study (Cantor et al. 1995) from the United


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States also reported a statistically significant increased risk for breast cancer; [however,

exposure was assigned based only on occupation listed on the death certificate]. No

increased risk of breast cancer was shown in the industry-based cohort studies. [The body

of literature from the occupational cohort studies has limited power to detect an effect.

The studies from the styrene-butadiene rubber and the styrene monomer and polymer

industries have been of men (except for Frentzel-Beyme et al. 1978, which did not state

the sex of the population, did not report a risk estimate for breast cancer, and was limited

by small numbers of expected (18.5) and observed cases (12) of malignant tumors).

Studies by Ruder et al. (2004), Wong et al. (1994), Kogevinas et al. (1994a), and Antilla

et al. (1998) included women; however, they were limited by small numbers of expected

and observed cases of breast cancer mortality (Ruder et al., Kogevinas et al. or low levels

of styrene exposure (Wong et al.). It seems reasonable that women are more likely to

have low-exposure jobs, and Kolstad et al. (1993, 1994) omitted females from

subsequent studies because the majority were not involved in the production of reinforced

plastics.

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[The cohort studies have not attempted to control for confounding factors that affect

breast cancer risk (such as body mass index, family history of breast cancer, alcohol use,

menopausal status, parity, hormone use, and age at first birth). In addition, cohort studies

often do not have sufficient follow-up time to detect effects on incidence or mortality

because of the long latency (sometimes in excess of 40 years) from the initiation to

detection of breast cancer. A decreased risk of breast cancer was found among women in

the cohort study of environmental exposure to styrene, but this study was limited by

small numbers of expected (7) and observed (4) deaths from breast cancer, and

questionable completeness of follow-up and identification of death certificates (Loughlin

et al. 1999). Note that a marginally significant increase in the incident risk ratio for breast

cancer was observed among a cohort of women army personnel in occupations with

medium to high potential exposure to volatile organic compounds (VOC), including

potential styrene exposure, (IRR = 1.48, 95% CI = 1.01 to 2.07 [95% CI = 1.03 to 2.12

also reported in a table]) compared with women with no or low VOC exposure (Rennix et

al. 2005), but no specific inferences for styrene can be drawn from this study.]


Table 3-9. Mortality or incidence of selected cancers among all workers in the reinforced-plastics industry Washington state

(Ruder et al.) United States

(Wong et al. 1994) Denmark

(Kolstad et al. 1994, 1995) Europea

(Kogevinas et al. 1994a)

SMR 95% CI Obs Exp SMR 95% CI Obs Exp SIR 95% CI Obs Exp SMR 95% CI Obs Exp Total

Obs/Exp

esophagus 2.30 1.19–4.02 12 5.2 1.92 1.05–3.22 14 7.3 0.92 0.50–1.57 13 14.2 0.82 0.47–1.31 17/12 20.9/16.0 51/42.7 pancreas 1.43 0.78–2.41 14 9.8 1.13 0.68–1.77 19 16.8 1.20 0.86–1.63 41 34.2 1.00 0.71–1.38 37/21 36.9/26.5 95/87.3 larynx NR NR NR NR 1.02 0.28–2.61 4 3.9 1.10 0.71–1.63 25 22.6 1.11 0.53–2.05 10/7 9.0/6.2 36/32.7 lung 1.14 0.90–1.43 76 66.7 1.41 1.20–1.64 162 115.2 1.12 0.98–1.26 248 222.4 0.99 0.87–1.13 235/168 237.3/175.0 654/579.3 all LH 0.74 0.42–1.20 16 21.6 0.82 0.56–1.17 31 37.7 1.20 0.98–1.44 112 93.7 0.93 0.71–1.20 60/37 64.4/46.2 196/199.2 lymphoma 0.39 0.01–2.19 1 2.6 0.72 0.20–1.85 4 5.5 1.33 0.96–1.80 42 31.5 0.77 0.43–1.28 15/11 19.4/14.2 58/53.8 Hodgkin’s disease 0.61 0.02–3.40 1 1.6 0.90 0.25–2.30 4 4.5 1.08 0.62–1.76 16 14.8 0.90 0.36–1.84 7/6 7.8/5.9

27/26.8

multiple myeloma NR NR NR NR NR NR NR NR 0.99 0.51–1.73 12 12.1 0.99 0.48–1.83 10/5 10.1/7.5

17/19.6

leukemia 0.60 0.19–1.40 5 8.3 0.74 0.37–1.33 11 14.8 1.22 0.88–1.65 42 34.4 1.04 0.69–1.50 28/15 27.0/18.6 73/76.1 Note that caveats regarding the pooling of data across cohort studies are discussed in Section 3.8, above. aThe number of expected and observed cases after the Danish male workers were excluded in the European study is presented after the slash (/). These numbers were used to pool the total number of observed and expected cases across the four studies to prevent any overlap between the Danish population and the European population. “≥” is used because for some cancer sites, the pooled number of expected cases was a slight underestimate because the expected number of cases was not given for all studies reporting observed number of cases.

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Table 3-10. Mortality or incidence of selected cancers among workers in high-styrene–exposure groups (laminators and others)* in the reinforced-plastics industry

Washington Statea

(Okun et al., Ruder et al.) United Statesb

(Wong, Wong et al.) Denmarkc

(Kolstad et al.) Europed

(Kogevinas et al.)

SMR 95% CI Obs Exp SMR 95% CI Obs Exp SIR 95% CI Obs Exp SMR 95% CI Obs Exp Total

Obs/Exp

esophagus 1.85 0.22–6.67 2 1.1 3.57 NR 2 0.6 NR NR NR NR 1.81 0.87–3.34 10 5.5 14/≥ 7.2 pancreas 1.88 0.51–4.81 4 2.1 0.80 NR 1 1.3 2.20 1.1–4.5 17 7.7 1.48 0.76–2.58 12 8.1 34/19.2 larynx NR NR NR NR 0.0 NR 0 NR NR NR NR NR 1.55 0.32–4.52 3 1.9 3/≥ 1.9 lung 1.29 0.76–2.04 18 14.0 0.90 NR 8 8.9 1.00 0.7–1.3 72 72.0 1.06 0.81–1.36 60 56.6 158/151.5 all LH 0.72 0.20–1.84 4 5.6 1.41 NR 4 2.8 1.09 0.74–1.55 31 28.4 0.81 0.43–1.39 13 16.0 52/52.8 lymphoma 0.0 NR 0 NR 2.55 NR 1 0.4 0.62 0.23–1.35 6 9.7 1.40 0.56–2.88 7 5.0 14/≥ 15.1 Hodgkin’s disease

1.78 0.05–9.89 1 0.6 0.00 NR 0 NR 1.41 0.57–2.91 7 5.0 1.33 0.27–3.88 3 2.3 11/≥ 7.9

multiple myeloma

NR NR NR NR NR NR NR NR 1.18 0.32–3.02 4 3.4 0.00 0.0–1.55 0 NR 4/≥ 3.4

leukemia 0.47 0.01–2.63 1 2.1 0.90 NR 1 1.1 1.38 0.75–2.32 14 10.1 0.48 0.10–1.39 3 6.3 19/19.6 * Note that in the Kolstad et al. studies, high-styrene exposure groups were defined as those who worked in plants where 50% to 100% of the workers were laminators. Note also that caveats regarding the pooling of data across cohort studies are discussed in Section 3.8, above. LH = lymphohematopoietic. aWorkers employed in fibrous glass or lamination departments. bOpen-mold process workers for more than two years. cAll workers employed in companies with 50% to 100% laminators. dLaminators, excluding the Danish workers included by Kolstad et al.


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3.9 Summary 1

Numerous epidemiological studies have evaluated the relationship between styrene and

cancer in humans. Most of the studies are cohort studies of workers in three major

industries: (1) the reinforced-plastics industry, (2) the styrene-butadiene rubber industry,

and (3) the styrene monomer and polymer industry. Two additional cohort studies (one

on biomonitored workers, and the second on environmental exposure to styrene-

butadiene), several case-control studies, and an ecological study have also been

published.

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The limitations of these studies include potential misclassification of styrene exposure

and disease, small numbers of long-term workers, inadequate follow-up, and the potential

for co-exposure to other chemicals. Thus, although more than a hundred thousand

workers have been studied to assess a possible carcinogenic effect of styrene exposure,

only a small fraction of well-characterized, high-level, and long-term styrene-exposed

workers have been followed for a sufficiently long time. In addition, most of the available

studies of occupational cohorts have focused only on male workers (who constitute the

majority of exposed workers) or have not performed gender-specific risk analyses. [Thus,

comparatively few data are available on cancer incidence or mortality among exposed

female workers, limiting the ability to evaluate breast cancer or cancers at tissue sites

specific for females.]

Workers in the reinforced-plastics industry have the highest levels of exposure and few

other potentially carcinogenic exposures, but many of the workers in this industry have

short-term exposure, often of less than a year. Cancer mortality or incidence was studied

in the following four populations of reinforced-plastics workers: (1) in Washington state

in the United States (Ruder et al. 2004), (2) in 30 manufacturing plants in unspecified

U.S. locations (Wong et al. 1994), (3) in Denmark (Kolstad et al. 1994), and (4) in

Europe (Denmark, Finland, Italy, Norway, United Kingdom, and Sweden) (Kogevinas et

al. 1994a). (The Danish and the European populations were partly overlapping, as 13,682

Danish male workers were included among the 36,610 male workers making up the

European cohort.)


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In the styrene-butadiene industry, the cohort studies are among the largest, with the

longest follow-up times. The principal methodological challenge is to separate the

potentially independent or synergistic effects of butadiene, a known human carcinogen,

which is highly correlated with styrene in this industry. Two independent (non-

overlapping populations) are available, a small cohort of 6,678 male workers at a rubber

tire manufacturing plant (a subset of the workers were engaged in the production of

styrene-butadiene and other rubbers) (McMichael et al. 1976a) and a larger cohort

established by Delzell and colleagues (Delzell et al. 1996, 2006) of 13,130 to 16,610

styrene-butadiene rubber industry workers from multiple plants in the United States and

Canada. The cohort established by Delzell includes most (but not all) of the workers from

two cohorts ― a 2-plant cohort (Texas) (Meinhardt et al. 1982) and an 8-plant cohort

originally established by Matanoski and colleagues (United States and Canada) and

reported in a series of previous publications (7 of the 8 plants were included in the

Delzell cohort). Thus, there is considerable overlap between these populations. Two

nested case-control studies (Matanoski et al.1997, Santos-Burgoa et al. 1992) of a single

group of cases with lymphohematopoietic cancers were available from the Matanoski

cohort. The Delzell cohort expanded the previous cohorts to include workers employed

from 1943 to January 1, 1991 and followed to 1998, whereas the earlier cohort included

workers employed until 1976 and followed until 1982. In addition, the individual study

populations were established by different procedures and exclusion criteria (which may

partly explain the lack of complete consistency in the number of study subjects across the

published studies) and often used different exposure assessments, selection of study

subjects, and types of analysis. Two types of analyses were conducted on the Delzell

cohort: external analyses reporting on standardized mortality ratios (SMRs) for the total

cohort or subsets of the cohorts for multiple cancers sites (Sathiakumar et al. 1998,

2005), and, secondly, internal analyses of relative risk (RR) estimates for quantitative

exposure to styrene and lymphohematopoietic cancers (Delzell et al. 2001, 2006,

Macaluso et al. 2006, Graff et al. 2005). (Dimethyldithiocarbamate [DMDTC] was also

included as a potential confounder in some analyses of lymphohematopoietic cancer in

the Delzell cohort, according to the authors, because of its potential immunosuppressant

activity in CD4+ lymphocytes, although its carcinogenicity has not been evaluated

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outside of this series of studies). Workers in the styrene monomer and polymer industry

may be exposed to a variety of chemicals, including benzene, toluene, ethylbenzene, and

various solvents, and the cohorts are smaller, with many short-term workers, and few

cancer outcomes.

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The potential effect of styrene on lymphohematopoietic cancers has been studied most

extensively. Findings for lymphohematopoietic cancer and other tumor sites of interest

are discussed below.

Lymphohematopoietic cancers

Statistically significant increases were observed for all lymphohematopoietic cancers

combined and leukemia among rubber-tire manufacturing workers (McMichael et al.

1976) and statistically nonsignificant increases were observed for combined

lymphohematopoietic cancers and some specific lymphohematopoietic cancers in the

Meinhardt and Matanoski cohorts, but the potentially confounding effects of butadiene

and other exposures were not analyzed. Two nested case-control studies (using different

types of analyses and exposure assessments and the same group of cases) from the

Matanoski cohort attempted to evaluate the relative contribution of styrene and butadiene

to lymphohematopoietic cancer mortality. Santos-Burgoa et al. (1992) found no

significant excess risks for combined and specific lymphohematopoietic cancers and

mean exposure after controlling for butadiene exposure. Matanoski et al. (1997)

calculated risks for both average and cumulative exposure to styrene. Taking into account

butadiene exposure, and demographic and employment variables in step-down regression

analyses, these models found, for an average exposure of 1 ppm vs. no exposure,

significant associations for all lymphohematopoietic cancers combined, lymphomas, and

myeloma, but not leukemia. For cumulative exposure, significant positive associations

between styrene exposure and combined lymphohematopoietic cancers, leukemia, and

myeloma were found, with butadiene exposure dropping out of each of the final models

except for leukemia.

Specific lymphohematopoietic cancers have been studied more extensively in the Delzell

cohort. With respect to leukemia, statistically significant increases have been reported


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among subgroups of workers with longer durations of employment and longer latency,

with the highest cumulative exposure, and in certain specific job groups (Sathiakumar et

al. 2005, Delzell et al. 2006). Internal analyses by Delzell et al. involving single-

chemical (styrene only), 2-chemical (styrene and butadiene), and 3-chemical (styrene,

butadiene, and DMDTC) models of cumulative exposure have shown increased relative

risks of leukemia with increasing cumulative styrene exposure. However, the response

was attenuated when controlling for exposure to butadiene and was no longer apparent

(RRs were less than or equal to one) after additionally controlling for DMDTC. Elevated

risks for leukemia were also observed with increasing exposure to styrene peaks in

single-chemical, 2-chemical and 3-chemical models (although it was attenuated

somewhat in the 2- and 3-chemical models) (Graff et al. 2005, Delzell et al. 2006).

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No statistically significant increased risks were found for other lymphohematopoietic

cancers in all employees of the Delzell cohort, but statistically significant risks of NHL

and CLL combined were found among workers with higher exposure in an external

(SMR) analysis, and in internal analyses among ever-hourly workers, ever-hourly

workers with 10+ years of employment and 20 to 29 years or 30 years since first hire, and

among specific job groups. Risks of NHL or NHL and CLL combined appeared to

increase with increasing cumulative styrene exposure; the risks increased when butadiene

was added to the model, and were somewhat attenuated in models that included DMDTC.

Exposure to butadiene did not appear to be related to NHL and CLL combined or NHL

risk. [However, it should be noted that no trend analyses were performed on these data.]

(Graff et al. 2005, Delzell et al. 2006). No associations were found for other types of

lymphohematopoietic cancers and styrene exposure in the Delzell cohort.

In the reinforced-plastics industry, among the highest-exposure groups, the total number

of observed versus expected deaths or cases across the four cohorts were comparable for

all lymphohematopoietic (52 observed vs. 52.8 expected), lymphomas (14 vs. 15.1), or

leukemia (19 vs. 19.8), and were slightly higher than expected for Hodgkin’s disease (11

observed vs. 7.9 expected) and multiple myeloma (4 vs. 3.4). Significantly increased

risks for leukemia incidence were reported in the Danish study among workers with

earlier first date of exposure, and who had worked at least 10 years since first


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employment, but not for workers employed for 1 year or more (Kolstad et al. 1994). In

the European multi-country cohort (which overlaps with the Danish study), no excess of

leukemia mortality was found, and no exposure-response relationships with cumulative

or average exposure were observed, although a non-significant trend was observed with

time since first exposure (Kogevinas et al. 1994a). With respect to other

lymphohematopoietic cancers, non-significantly increased risks for non-Hodgkin’s

lymphoma were found in the Danish and European multi-country cohorts. Positive

exposure-response relationships with average styrene exposure and time since first

exposure was observed for lymphohematopoietic cancers (P = 0.019 and 0.012,

respectively) and for malignant lymphoma (P = 0.052 and 0. 072, respectively) in the

European multi-country cohort, but no relationship with cumulative exposure was

observed (Kogevinas et al. 1994a). No excesses in mortality from any

lymphohematopoietic cancers were observed in the two smaller cohort studies (Ruder et

al. 2004 and Wong et al. 1994). In the styrene monomer and polymer industries, the risk

of lymphohematopoietic malignancies was also increased in most of the studies (as well

as the total number of observed cases across studies), but these workers might also have

been exposed to benzene.

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Pancreatic cancer

Among the highest styrene-exposed group in the reinforced-plastics industry, there was

an excess in the total number of observed cases of pancreatic cancer across the four

cohort studies compared with the total number of expected cases [corresponding to an

SMR of 1.77 (95 % CI = 1.23 to 247)]. Increases in pancreatic cancer risk were observed

in three of the four reinforced-plastics industry cohorts (one of which was statistically

significant [Kolstad et al. 1995], and the other two of which were nonsignificant

[Kogevinas et al. 1994a, Ruder et al. 2004]). The risk of pancreatic cancer was slightly

higher among the Danish workers with longer term employment and earlier start date,

and increased with cumulative exposure in the multi-plant cohort. No indications of

exposure-response relationships were found in the smaller U.S. cohorts. Statistically

nonsignificant increased risks were also observed in one study in the styrene monomer

and polymer industry (Frentzel-Beyme et al. 1978), and among biomonitored workers


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(10 years after the first measurement) (Anttila et al. 1998). However, no increased risk of

pancreatic cancer was reported among styrene-butadiene workers (Sathiakumar et al.

2005).

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Esophageal cancer

Among workers with high potential exposure to styrene, increases in esophageal cancer

risk were reported in three of the four cohorts (statistically significant increases in

mortality were observed among all exposed workers in the two U.S. studies of

reinforced-plastics workers [Ruder et al. 2004, and Wong et al. 1994] and a statistically

nonsignificant increase among a subset of laminators in the European cohort [Kogevinas

et al. 1994a]). Risks were not elevated among the Danish reinforced-plastics workers

(Kolstad et al. 1994). Across the industry, an approximately 2-fold excess of esophageal

cancer was observed among high-exposed groups (laminators and others). A

nonsignificant trend with cumulative exposure was reported in the European multi-

country study. No increases in risk were reported among styrene-butadiene rubber

workers or among styrene monomer and polymer workers.

Other sites

Findings were less consistent for cancer at other sites. Significantly increased risks were

observed for cancers of the lung, larynx, stomach, benign neoplasms, cervix and other

female tumors, prostate, rectum, and urinary system in either a single study or two

studies. There were some supporting exposure-response data for cancers of the urinary

system and rectum. A significant increase in breast cancer mortality was observed in a

case-control study of occupational exposures among adult females (Cantor et al. 1995),

although there was no evidence of increased risk between low- and high-exposure

categories. An ecological study reported a significant increase in the risk of invasive

breast cancer in the general population, but exposure estimates were based on

environmental releases of styrene, which are the least precise measures of exposure.



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4 Studies of Cancer in Experimental Animals 1

The carcinogenicity of styrene has been investigated in experimental animals (primarily

mice and rats) by several routes of administration, and IARC (1994a, 2002) has evaluated

the carcinogenicity of styrene. The 1994 IARC review included four studies in mice

(three gavage and one intraperitoneal [i.p.] injection study) and seven studies in rats

(three gavage, one drinking water, one inhalation, one i.p., and one subcutaneous (s.c.)

injection study) and concluded that there was limited evidence in experimental animals

for the carcinogenicity of styrene. IARC (2002) also concluded that there was limited

evidence in experimental animals for the carcinogenicity of styrene. The latter review

included two inhalation studies (one in mice and one in rats) that were not available for

the previous review, and the IARC working group considered the earlier gavage studies

in mice as inadequate.

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The data and findings from the publicly available, peer-reviewed carcinogenicity studies

of styrene in experimental animals are summarized in this section. This includes the

studies reviewed by WHO (1983), Huff (1984), Bond (1989), McConnell and Swenberg

(1993, 1994), and IARC (1994a, 2002). In addition, information from one unpublished

study (Jersey et al. (1978), a two-year inhalation study in rats conducted by Dow

Chemical), is included based on reviews by WHO (1983), Huff (1984), McConnell and

Swenberg (1993, 1994), and Cohen et al. (2002)5.

Section 4.1 presents carcinogenicity data for mice, and Section 4.2 presents data for rats.

These sections are organized by route of administration. Section 4.3 includes data from

one carcinogenicity study with a mixture containing styrene and β-nitrostyrene, and

Section 4.4 briefly reviews carcinogenicity data for styrene-7,8-oxide, the primary

metabolite of styrene. All the data are summarized in Section 4.5.

4.1 Mice 25 Two oral studies (NCI 1979a, Ponomarkov and Tomatis 1978), one inhalation study

(Cruzan et al. 2001), and one i.p. study (Brunnemann et al. 1992) are reviewed below.



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The Ponomarkov and Tomatis (1978) study included two strains of mice and included

both pre- and postnatal exposure.

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4.1.1 Oral 3 Styrene [one impurity (0.3%) was reported in 1 of the 6 batches purchased for the

bioassay, but purity was not specified for the remaining 5 batches] was administered by

gavage in corn oil to groups of 50 male and 50 female B6C3F1 mice for 78 weeks (NCI

1979a). The mice were approximately 6 weeks old at the beginning of the study. Test

groups received styrene at 150 or 300 mg/kg b.w., 5 days per week, while control groups

of 20 male and 20 female mice were exposed to corn oil alone. Mice were held for an

additional 13 weeks after the last treatment. There was a slight dose-related body weight

depression in female mice. Survival in male mice was 78% (high dose), 92% (low dose),

and 100% (controls); survival in female mice was 76% (high dose), 80% (low dose), and

90% (controls). The Tarone test for dose-related mortality was significant in male mice

(P = 0.003). Therefore, animals that did not survive at least 52 weeks or died before the

first appearance of the tumor(s) of interest were not included in the analysis. The

Cochran-Armitage exact trend analysis also indicated a significant dose-response

relationship for combined alveolar/bronchiolar neoplasms in male mice. This was

supported by an increased incidence of alveolar/bronchiolar neoplasms (adenoma and

carcinoma combined) in male mice in the high-dose group compared with controls (Table

4-1). Because the incidence of lung tumors in the male vehicle-treated controls (0%) in

this study was unusually low compared with historical untreated controls (32 of 271,

12%), there was some uncertainty regarding the significance of the lung tumors. NCI

(1979a) reported that the historical incidence of these tumors in vehicle control male

mice was 0 of 40 (2 studies from Litton Bionetics, including the styrene study); however,

this was considered by NCI to be too small a number of animals for meaningful use as

historical controls. [The NTP reviewed (for the purpose of this document) lung tumor

incidences in historical vehicle controls from NCI studies conducted at other laboratories.

However, although the studies were performed at different laboratories, the historical

vehicle control animals were from the same source and same study protocol, and the tests

were performed in the same chronological window. The selection criteria included data

for corn oil vehicle controls for gavage studies in male B6C3F1 mice conducted prior to


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1979 with a similar duration (total of 91 weeks) and from the same source as the styrene

study. In addition to the two studies from Litton Bionetics (NTP 1979a, 1979b), there

were 12 applicable studies conducted by Hazleton Laboratories (NTP 1976a, 1976b,

1977, 1978a, 1978b, 1978c, 1978d, 1978e, 1978f, 1978g, 1978h, 1978i). The incidence of

combined lung tumors in historical vehicle controls from these 14 studies was 11 of 273

(4%). Therefore, the incidence of lung tumors in control male mice in the NCI (1979a)

study was not unusually low and support the finding that lung tumors as a result of

styrene exposure are statistically significant.] In addition to the lung tumors,

hepatocellular carcinomas were reported in male mice but occurred at a higher incidence

in controls (20%) than in the treatment groups (6% to 14%). Thus, there were no

significant hepatocellular tumor findings in male mice. No other tumors were considered

as dose-related. Although there was a significant trend for hepatocellular adenoma (P =

0.03) in female mice, there were no hepatocellular carcinomas in any female mice, and

the NCI did not consider this marginal adenoma effect to be related to styrene. NCI

(1979a) concluded that there was suggestive evidence for the carcinogenicity of styrene

in male B6C3F1 mice, but no convincing evidence was obtained for either sex.

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Groups of pregnant O20 (a strain susceptible to lung tumors) and C57Bl mice were

administered a single dose of styrene (O20 mice, 1,350 mg/kg; C57Bl mice, 300 mg/kg)

[the rationale for choosing these doses was not discussed], dissolved in olive oil, or a

single dose of olive oil (vehicle control) by gavage on gestation day 17 (Ponomarkov and

Tomatis 1978). The purity of styrene used in this study was > 99%. After weaning, their

progeny were administered the same dose of styrene or olive oil once per week. Separate

groups received no treatment and served as untreated controls. Styrene treatment of O20

mice was suspended after 16 weeks because of toxicity, while C57Bl mice received

weekly treatments until their deaths or 120 weeks. Litter sizes were similar in all groups

except in the C57Bl vehicle control group, which had less than one-half the number of

animals in the other study groups. Preweaning mortality was higher in the styrene-treated

group of O20 mice (43%) compared with the vehicle control group (22%). Mortality

remained high in O20 mice after styrene treatment was suspended at 16 weeks; however,

body weights were similar in all groups. Mortality was not increased in C57Bl mice

treated with styrene.


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Table 4-1. Tumor incidences in B6C3F1 mice exposed to styrene by gavage for 78 weeks and surviving for at least 52 weeks

Tumor incidence [%]

Alveolar/bronchiolar

Sex Dose

(mg/kg)

Initial no.

mice Hepatocellular

adenoma Adenoma Carcinoma Combined

Male 0 150 300 [Trenda]

20 50 50

1/20 [5] 5/47 [10.6] 7/43 [16.3]

[NS]

0/20 [0] 3/44 [6.8] 4/43 [9.3]

[NS]

0/20 [0] 3/44 [6.8]

5/43 [11.6] [P = 0.08]

0/20 [0]b 6/44 [13.6]

9/43 [20.9]* [P = 0.02]

Female 0 150 300 [Trenda]

20 50 50

0/20 [0] 1/44 [2.3]

5/43 [11.6[ [P = 0.03]

0/20 [0] 1/43 [2.3] 3/43 [7.0]

[NS]

0/20 [0] 0/43 [0] 0/43 [0]

[NS]

0/20 [0] 1/43 [2.3] 3/43 [7.0]

[NS] Source: NCI 1979a. NS = not significant. * P < 0.05 (compared with concurrent controls, one-tailed Fisher’s exact test). a[Cochran-Armitage exact test for positive dose-response trend performed by NTP]. b Incidences in untreated historical controls were 32/271 or 12% [reported by NCI 1979a] and in vehicle controls were 11/273 or 4% [calculated by NTP for the present report].

Tumor incidences are shown in Tables 4-2a for O20 mice and 4-2b for C57Bl mice.

There was a statistically significant (P < 0.01) increased incidence of total lung tumors in

both male and female O20 mice treated with styrene compared with the vehicle control

groups. [When compared with the untreated groups, the difference was statistically

significant (P < 0.001) only for the females.] Lung tumors were reported to occur at an

earlier age in the styrene-treated progeny than in control progeny, [but this may be the

result of higher mortality in the styrene-treated mice rather than an effect of styrene.

Information necessary to interpret the significance of this observation (whether the lung

tumors were incidental or fatal) was not reported.] The authors noted that this study had

severe limitations because of the severe toxicity and early mortality in O20 mice but

concluded that there was weak evidence for the carcinogenicity of styrene in O20 mice

when administered at a high dose level.

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The predominant tumors occurring in C57Bl mice included lymphoma and lung or liver

tumors (Table 4-2b). The incidences of these tumors in styrene-treated mice (dams or

progeny) were not significantly higher than controls. While the authors reported that the

higher incidence of liver tumors in styrene-treated male mice (3 carcinomas; 12.5%) was


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cause for some concern, one adenoma was also observed in a vehicle control male (8.3%)

and one adenoma was observed in an untreated control male (2.1%).

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Table 4-2a. Lung tumor incidences in O20 mice exposed to styrene in utero and weekly by gavage for 16 weeks after weaning

Lung tumor incidence (%)a

Treatment Group Initial no.

mice Adenoma Carcinoma Total Untreated Male

Female 54 47

22/53 (41.5) 11/47 (23.4)

12/53 (22.6) 14/47 (29.8)

34/53 (64.2) 25/47 (53.2)

Vehicle control

Damsb Progeny

Male Female

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20 22

1/8 (12.5) 4/19 (21.1) 10/21 (47.6)

4/8 (50) 4/19 (21.1) 4/21 (19.0)

5/8 (62.5) 8/19 (42.1) 14/21 (66.7)

Styrene (1,350 mg/kg)

Damsb Progeny

Male Female

29

45 39

4/20 (20) 12/23 (52.2) 14/32 (43.8)

7/20 (35) 8/23 (34.8) 18/32 (56.3)

11/20 (55) 20/23 (87)** 32/32 (100)**c

Source: Ponomarkov and Tomatis 1978. ** P < 0.01 compared with vehicle control-treated group significance levels reported only for total tumors; statistical test not reported]. a Based on the number of animals surviving until the time the first tumor was observed. b On gestational day 17, dams received a single dose by gavage of 1,350 mg/kg; after weaning, progeny received weekly doses of 1,350 mg/kg. c P < 0.001 when female progeny of styrene-treated dams compared with untreated females, and the authors reported a non-significant difference for males [statistical method not reported]; [NTP calculated P = 0.037 by Fisher’s exact test for male progeny of styrene-treated dams compared with untreated males.]

Table 4-2b. Tumor incidences in C57Bl mice exposed to styrene in utero and weekly by gavage for 120 weeks after weaning

Tumor incidence (%)a

Treatment Group Initial

no. mice Lymphoma Lung Liverb Other Untreated Male

Female 51 49

13/47 (27.7) 20/47 (42.5)

5/47 (10.6) 1/47 (2.1)

1/47 (2.1) 0/47 (0)

3/47 (6.4)c 4/47 (8.5)d

Vehicle control

Damse Progeny

Male Female

5

12 13

3/5 (60.0) 3/12 (25.0) 5/13 (38.4)

0/5 (0) 3/12 (25.0) 1/13 (7.7)

0/5 (0) 1/12 (8.3) 0/13 (0)

2/5 (40.0)f 2/12 (16.7)g 1/13 (7.7)h

Styrene (300 mg/kg)

Damse Progeny

Male Female

15

27 27

10/12 (83.3) 9/24 (37.5) 13/24 (54.2)

1/12 (8.3) 1/24 (4.2) 1/24 (4.2)

0/12 (0) 3/24 (12.5) 0/24 (0)

3/12 (25.0)i 1/24 (4.2)j 4/24 (16.7)k

Source: Ponomarkov and Tomatis 1978. a Based on the number of animals surviving until the time the first tumor was observed. bLiver tumors in males in the 2 control groups were adenomas; and in the styrene-treated group were carcinomas. c Forestomach papilloma, duodenum polyp, kidney adenocarcinoma. d Uterine sarcoma (2), lacrimal gland adenoma, ovary theca-cell tumor.


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e On gestational day 17, dams received a single dose by gavage of 300 mg/kg; after weaning, progeny received weekly doses of 300 mg/kg. f Pituitary adenoma. gHemangioendothelioma of the leg, hemangioma (s.c.). hHemangioma (s.c.). i Jaw osteosarcoma, ovary granulosa-cell tumor, pituitary adenoma. j Urinary bladder papilloma. k Uterine sarcoma (2), adenoma of the lacrimal gland, ovary theca-cell tumor.

4.1.2 Inhalation 1 Cruzan et al. (2001) exposed groups of 70 male and 70 female CD-1 mice to styrene

vapor (whole-body exposure) at concentrations of 0, 20, 40, 80, or 160 ppm, 6 hours/day,

5 days/week for 98 (females) or 104 weeks (males). The purity of styrene used in this

study was 99.5% to 99.8%. The mice were 4 weeks old when received. Interim sacrifices

of 10 animals per sex per group were conducted after 52 and 78 weeks. Styrene exposure

did not affect survival in male mice, and apart from two deaths in the 160-ppm group

during the first 2 weeks, survival was slightly increased in styrene-exposed female mice.

Body weight gains were significantly less in the high-dose groups of both sexes during

the first 13 weeks of the study. At the end of the study, males (80 and 160 ppm) and

females (160 ppm) gained significantly less weight than controls.

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There was an increased incidence of benign lung tumors (alveolar/bronchiolar adenomas)

at several exposure levels in both sexes and malignant lung tumors (alveolar/bronchiolar

carcinomas) in high-exposure female mice at the end of the study (Table 4-3). Incidences

of adenomas and carcinomas combined were not evaluated by the study authors, but

Cohen et al. (2002) reported that the combined tumor incidences were significantly

higher than controls at exposures of 40, 80, and 160 ppm (male mice) and 20, 40, and 160

ppm (female mice). [These results for combined tumor incidences were confirmed by

NTP by Fisher’s exact test for pairwise comparisons and Cochran-Armitage exact trend

test (see Table 4-3).] Cruzan et al. (2001) also reported that there was a significant

positive trend for benign lung tumors in both sexes and for benign plus malignant tumors

in female mice. The incidence of lung tumors was not increased in styrene-exposed mice

sacrificed at 52 or 78 weeks.


Table 4-3. Lung tumor incidence in CD-1 mice exposed to styrene by inhalation for 98 or 104 weeksb

Alveolar/bronchiolar tumor incidencea [%] Sex

Exposure conc (ppm) Adenoma Carcinoma Combined

Male 0 20 40 80 160

[Trend]

15/50 [30] 21/50 [42]

35/50 [70]**** c 30/50 [60]** c 33/50 [66]*** c [P < 0.001]

4/50 [8] 5/50 [10] 3/50 [6] 6/50 [12] 7/50 [14] [NS]

17/50 [34] 24/50 [48] 36/50 [72]*** 30/50 [60]** 36/50 [72]*** [P < 0.001]

Female 0 20 40 80 160

[Trend]

6/50 [12] 16/50 [32]* c 16/50 [32]* c

11/50 [22] 24/50 [48]**** c

[P < 0.001]

0/50 [0] 0/50 [0]

2/50 [4] 0/50 [0]

7/50 [14]** c [P < 0.001]

6/50 [12] 16/50 [32]* 17/50 [34]** 11/50 [22] 27/50 [54]**** [P < 0.0001]

Source: Cruzan et al. 2001; Cohen et al. 2002. NS = not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, [Fisher’s exact test for pairwise comparisons and the Cochran-Armitage exact trend test conducted by NTP.] a(Number of mice with tumor) / (number of animals examined for each tissue type). bDue to high mortality, females in this study were terminated early at 98 weeks; males were exposed until planned study termination at 104 weeks. c Reported by Cruzan et al. 2001 as P < 0.05. .

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4.1.3 Intraperitoneal injection 1 In a screening study, Brunnemann et al. (1992) exposed 25 female A/J mice (a strain

susceptible to lung tumors) to a total dose of 200 μmol [~100 mg/kg b.w.] styrene (>

99% purity) given by i.p. injection three times per week for 20 doses. This study also

included a positive control group exposed to 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-

butanone (NNK). The mice were 6 to 8 weeks old at the beginning of the study. Test

animals were held for 20 weeks after the last injection and examined for lung tumors.

Three mice exposed to styrene developed lung adenoma compared with one in the control

group. The difference was not significant, and the authors concluded that styrene was not

tumorigenic under the conditions of this bioassay. [The short duration of the study, single

sex, and small group size limit this study as a test for carcinogenic activity.]

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4.2 Rats 12 The carcinogenicity of styrene in rats has been investigated following oral administration

(Beliles et al. 1985, Conti et al. 1988, Maltoni et al. 1982, NCI 1979a, Ponomarkov and

Tomatis 1978), inhalation (Conti et al. 1988, Cruzan et al. 1998, Jersey et al. 1978), and

i.p. and s.c. injection (Conti et al. 1988). These studies are reviewed in the following

sections.

4.2.1 Oral 18 Styrene was administered by gavage in three studies and in the drinking water in one

study. These studies are reviewed briefly below and the results are summarized in Table

4-5.

Ponomarkov and Tomatis (1978) investigated the carcinogenic effects of prenatal and

postnatal exposure to styrene (purity 99%). On the 17th day of gestation, 21 pregnant BD

IV rats received a single oral dose of 1,350 mg/kg styrene dissolved in olive oil. After

weaning, the offspring (73 males and 71 females) were given weekly doses of 500 mg/kg

by stomach tube throughout their lifespan (all survivors were killed at 120 weeks) [Only

one treatment group was used and dosing was only once per week.] The control groups

were similarly treated with olive oil. Litter sizes were not affected by styrene treatment,

and no differences in survival or body weights were noted. The incidences of tumors in

the styrene-treated rats were not significantly higher than those of controls. The authors


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reported that stomach tumors observed in one styrene-treated dam and two styrene-

treated female progeny were of “some concern” because they were “rarely seen in

controls.” While the histologic types of these stomach tumors were not specified in the

table, they were described in the text as an adenoma, a fibrosarcoma, and a

carcinosarcoma without specific attribution to a particular dose group. A stomach

fibrosarcoma was observed in one of the vehicle control female progeny. [The low

incidence of stomach tumors and inadequate reporting of tumor types limits concluding

that these tumors are associated with treatment.]

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NCI (1979a) treated groups of 50 male and female F344 rats with 500, 1,000, or 2,000

mg/kg styrene (purity described in Section 4.1.1) in corn oil by gavage 5 days per week

for 78 weeks (mid- and high-dose groups). The rats were 6 weeks old at the beginning of

the study. Surviving animals were sacrificed after 104 or 105 weeks. Only 6 of 50 male

rats in the high-dose group survived past week 53, and only 7 of 50 female rats survived

past week 70. Because of poor survival, the high-dose groups were not included in the

statistical analysis of tumors. Due to excessive mortality in the high- and medium-dose

groups, additional groups of male and female rats were placed on test in week 23. These

dosed rats were intubated with styrene at a level of 500 mg/kg for 103 weeks, followed

by a 1-week observation period, when no test chemicals were used. Separate vehicle

controls were also started for this group. Survival of low- (44/50) and medium-dose rats

(47/50) at week 90 was considered adequate by the study authors. No increased tumor

incidences were observed in any of the treatment groups.

Conti et al. (1988) investigated the long-term carcinogenicity of styrene (purity 99.8%) in

Sprague-Dawley rats. A previous publication from this study focused only on brain

tumors (Maltoni et al. 1982), while the complete results were reported by Conti et al.

Groups of 40 male and female rats (13 weeks old at the start of the experiment) were

exposed by stomach tube to 50 or 250 mg/kg styrene, 4 or 5 days per week for 52 weeks

and held until death [less than lifetime exposure duration, low doses]. The control group

received olive oil. Females in the high-dose group had a higher mortality rate compared

with controls. Body weight was not affected by styrene treatment; [however, there was

limited reporting of results]. No increased tumor incidences were reported.


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Sprague-Dawley rats (7 weeks old at the start of the study) were exposed to styrene

(purity ≥ 98.9%) in their drinking water for two years (Beliles et al. 1985). [This study

was identified as the Chemical Manufacturers Association study by Huff (1984) before it

was published.] Nominal concentrations were 125 or 250 ppm. The authors noted that the

calculated daily doses in this study (7.7 to 14 mg/kg in males and 12 to 20.5 mg/kg in

females) were at least an order of magnitude lower than doses used in other chronic oral

toxicity studies with styrene, such as the NCI study above (500, 1,000, or 2,000 mg/kg)

[solubility of styrene in water limited the dosage]. Chronic toxicity and reproductive

performance were evaluated. The test groups included 50 male and 70 female rats, while

the control group consisted of 76 males and 106 females. This study also evaluated the

effects of styrene on reproductive function through three generations (see Section 5.4.2

for reproductive toxicity). The only treatment-related effect identified was a decrease in

water consumption. There was no effect on mortality. The authors reported that all

tumors observed were either common, spontaneously occurring tumors of Sprague-

Dawley rats or were uncommon tumors that affected only individual rats in the treatment

groups and concluded that styrene administered in drinking water did not produce

deleterious dose-related effects in rats. Tumors were identified only by tissue (number of

tissues examined and total number of tumors) [actual tumor rates not reported]. However,

Huff et al. (1984) reexamined these data and reported specific mammary tumor

incidences for fibroadenoma, adenoma, adenocarcinoma, and combined mammary

tumors (Table 4-4). The authors reported marginal increase in combined mammary gland

tumors (fibroadenoma, adenoma, and adenocarcinoma) in female rats. Incidences were

49 of 96 (51%) in controls, 18 of 30 (60%) in the low-dose group and 40 of 60 (66.7%) in

the high-dose group. Huff reported that there was a significant dose-related trend (P =

0.032), and the incidence in the high-dose group was significantly higher than the control

group (P = 0.039, Fisher’s exact test).

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Table 4-4. Mammary gland tumor incidence in Sprague-Dawley rats exposed to styrene in drinking water for 104 weeks Exposure ppm (mg/kg) Mammary Gland Tumor Incidence (%)

Fibroadenoma Adenoma Adenocarcinoma Combined 0 45/96 (49) 1/96 (1) 8/96 (8) 49/96 (51) 125 (12) 15/30 (50) 0/30 (0) 5/30 (17) 18/30 (60) 250 (21) 37/60 (62)* 0/60 (0) 8/60 (13) 40/60 (67) [Trend] [P = 0.046] [NS] [NS] [–a] Source: Huff et al. 1984. NS = not significant. *P = 0.05 [Fisher’s exact test for pairwise comparison and Cochran-Armitage exact trend test P values calculated by NTP.] a [Statistics not reported by NTP for benign and malignant tumors combined because of lack of information on the histogenesis of the tumors.]


Table 4-5. Summary of carcinogenicity studies in rats exposed to styrene by oral administration Duration (wk)

Reference Strain Dose

(mg/kg)a No. ratsb Exposure Study Results/comments Ponomarkov and Tomatis 1978

BD IV 0 500c

36–39 71–73

120 120 No treatment-related increases of any type of tumor [Limitations: Only one treatment group, dosing schedule only once per week]

NCI 1979a F344 0 500 1000 2000

40 50 50 50

104–105 103 78 78

104–105 104 105 105

No treatment-related increases of any type of tumor [Limitations: High mortality in high-dose group (both sexes) resulted in an inadequate number of animals for statistical analyses]

Maltoni et al. 1982, Conti et al. 1988

Sprague-Dawley 0 50 250

40 40 40

52

Held until death

No increase in tumors [Limitations: High mortality rate in high-dose females, limited reporting, less than lifetime exposure duration]

Beliles et al. 1985 Sprague-Dawley Males 0 7.7d 14d

Females 0 12d 20.5d

76 50 50 106 70 70

104 104

104 104

No treatment-related increases of any type of tumor reported by study authors. [Limitations: Low doses]

a Administered by gavage 4 or 5 days per week unless otherwise noted. b Includes numbers for each sex unless otherwise noted. c Dams received a single dose of 1,350 mg/kg on gestation day 17. After weaning, progeny received weekly doses of 500 mg/kg for life or until study termination. d Administered in drinking water (125 or 250 ppm); conversion to mg/kg as reported by Beliles et al. (1985).

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4.2.2 Inhalation 1 Conti et al. (1988) exposed groups of 30 male and 30 female Sprague-Dawley rats to

styrene vapors (purity 99.8%) in stainless steel inhalation chambers at concentrations of

25, 50, 100, 200, or 300 ppm for 4 hours/day, 5 days/week for 52 weeks and held the

animals until death. The animals were 13 weeks old at the beginning of the study. The

control groups included 60 rats of each sex. There were no significant differences in body

weight or mortality between exposed and control groups. A higher incidence, but not

statistically significant, of total malignant tumors that was not due to an increase in any

specific tumor was observed in male (8 of 30, 26.7%) and female (13 of 30, 43.3%) rats

exposed to 100 ppm compared with controls (10 of 60, 16.7% in males and 16 of 60,

26.7% in females). Total malignant tumors were not increased at the two highest dose

levels. The incidence of malignant mammary tumors was higher in female rats (all

exposed groups) compared with controls (Table 4-6). [Therefore, it appears that the

reported incidence of malignant mammary tumors was too high, or the incidence of total

malignant tumors was too low.] The authors concluded that the increased incidence of

malignant mammary tumors in female rats was “treatment-related and statistically

significant” and that this study demonstrated a weak carcinogenic effect for styrene.

IARC (1994a) considered this study to be inconclusive because of incomplete reporting

and the high incidence of spontaneous mammary tumors.

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Table 4-6. Incidence of mammary tumors in Sprague-Dawley rats exposed to styrene by inhalation for 52 weeks

Mammary tumor incidencea (%)

Sex Exposure

conc. (ppm) Initial no. rats Benign +

malignantb Malignantc Male 0

25 50 100 200 300

[Trend]

60 30 30 30 30 30

8/60 (13.3) 6/30 (20.0) 3/30 (10.0) 6/30 (20.0) 4/30 (13.3) 5/30 (16.7)

[NRb]

1/60 (1.7) 1/30 (3.3) 1/30 (3.3)d

0/30 (0) 1/30 (3.3) 0/30 (0)

[NS] Female 0

25 50 100 200 300

[Trend]

60 30 30 30 30 30

34/60 (56.7) 24/30 (80.0) 21/30 (70.0) 23/30 (76.7) 24/30 (80.0) 25/30 (83.3)

[NRb]

6/60 (10.0) 6/30 (20.0) 4/30 (13.3) 9/30 (30.0)*

12/30 (40.0)e*** 9/30 (30.0)* [P = 0.002]

Source: Conti et al. 1988. * P < 0.05, ** P < 0.01, *** P < 0.001. [Table provides significance values calculated by NTP: Fisher’s exact test for pairwise comparison and Cochran-Armitage exact test for trend.] conc. = concentration, NR = not reported, NS = not significant. a (number mice with tumor) / (number of animals examined for each tissue type) b Authors noted higher incidence in all exposed groups of females compared with controls, but increases were not reported to be statistically significant and specific tumor types were not reported. [Statistics not reported for benign plus malignant tumors because of lack of information on the histogenesis of the tumors.] c Authors reported to be treatment-related and statistically significant for females; however, no specific dosed group(s) was identified. d Reported as 3% by Conti et al. e [Reported incidence may be in error because it exceeds the incidence reported for total malignant tumors of 10 of 30 (33.3%). When the Cochran-Armitage exact test was recalculated with 10/30 as the tumor incidence for the 200-ppm group of females, the P value was 0.004. Trend tests were performed by NTP.]

An unpublished study (Jersey et al. 1978) was reviewed by WHO (1983), Huff (1984)

[note that Huff referenced the paper as Dow 1978], ATSDR (1992), McConnell and

Swenberg (1993, 1994), and Cohen et al. (2002) and is included here based on

information from these secondary sources. [However, without the original data provided

in the unpublished laboratory report data essential to the interpretation of this study are

missing.] Groups of 96 or 97 male and 96 female Sprague-Dawley rats (7 to 8 weeks of

age) were exposed to 600- or 1,200-ppm styrene (purity 99.5%) for 6 hours/day, 5

days/week. After 2 months, the concentration for the high-dose group was reduced to

1,000 ppm because of excessive toxicity. Interim sacrifices of 5 or 6 animals of each sex

were conducted after 6 and 12 months. Styrene exposure was stopped after 18.3 months

in males and 20.7 months in females because mortality had reached 50%. Animals were

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observed until their deaths, or 24 months. Survival was lower in males than in females

(attributed to a high incidence of chronic murine pneumonia in males). At 24 months, the

number of surviving animals was as follows: control group (5 males and 30 females),

600-ppm group (18 males and 30 females), 1,000-ppm group (6 males and 22 females)

(Cohen et al. 2002). Although the incidence of mammary adenocarcinoma in females in

the 600-ppm group was 8.2% and was significantly higher than in controls, the authors

concluded that there was no causal association with styrene exposure because mammary

adenocarcinoma did not occur in the high-dose group, the incidence of mammary

adenocarcinoma in the control group (1.2%) was low compared with historical controls

(mean of 5.8%), and the range among historical controls (0% to 9%) contained the rate

observed in the treatment group. Incidences of mammary fibroadenoma showed no

evidence of a styrene effect (WHO 1983). Combined incidences of lymphosarcoma and

leukemia in female rats were 1.2% in controls and 7.1% in both exposed groups;

incidences in males were 1.2% in controls, 5.8% in the 600-ppm group, and 1.2% in the

1,000-ppm group (Table 4-7). Incidences of lymphosarcoma and leukemia were not

statistically significant compared with the concurrent controls but were significant when

compared with historical controls. [The combined incidence of leukemia and

lymphosarcoma in historical controls was not provided; however, these tumors are not

typically combined in carcinogenicity studies.] Huff (1984) mentioned that the authors

concluded that the data were “suggestive of an association between the exposure of these

female rats to styrene vapor and an increased incidence of tumors of the leukemia-

lymphosarcoma type. In males, the results are even more inconclusive but tend to support

the suggestive association found in the females.” McConnell and Swenberg (1994) noted

that “this study was seriously flawed by the presence of chronic murine pneumonia,

which caused a high rate of mortality in both control and exposed male rats; it was less a

factor in females.”

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Table 4-7. Mammary tumors and leukemia or lymphosarcoma in Sprague-Dawley rats exposed to styrene by inhalation for 18 to ~21 months

Tumor incidence (%)

Sex

Exposure conc

(ppm) Initial no.

ratsa Treatment

duration (mo)Mammary

adenocarcinomaLeukemia or

lymphosarcoma Male 0

600 1,000

[Trend] e

96 97 96

24 18.3 18.3

0/85 (0) 0/86 (0) 0/84 (0)

[NS]

1/85 (1.2) 5/86 (5.8) 1/84 (1.2)

[NS] Female 0

600 1,000b

[Trend] e

96 96 96

24 20.7 20.7

1/85 (1.2)c 7/85 (8.2)*

0/85 (0) [P = 0.002]

1/85 (1.2) 6/85 (7.1) d 6/85 (7.1) d

[P = 0.035] Source: unpublished study by Jersey et al. 1978, cited in Huff 1984; WHO 1983, McConnell and Swenberg 1993, 1994, Cohen et al. 2002. * Significantly different from the control, P value and statistical method not reported. [P = 0.032 by Fisher’s exact test calculated by NTP.] conc. = concentration, NS = not significant. a Interim sacrifice of 5 animals of each sex at 6 months and 6 animals of each sex at 12 months. b Initial concentration was 1,200 ppm for the first 2 months then decreased to 1,000 ppm because of toxicity. c Incidence in historical controls was 5.8%. d Reported to be significant when compared with historical controls (1.36% [11/808]; range = 0%–2.64%), but historical control data were not provided in a published report. e[Cochran-Armitage exact trend test conducted by NTP.]

Cruzan et al. (1998) exposed groups of 70 male and 70 female Sprague-Dawley rats to

styrene vapor (purity 99.5% to 99.7%) at 0, 50, 200, 500, or 1,000 ppm 6 hours/day, 5

days/week for 104 weeks in inhalation chambers. Surviving animals were killed during

weeks 105 to 107. Styrene exposure did not affect survival in males, but survival in

females in the 500- and 1,000-ppm groups was higher than in the control group. Eight

males in the 1,000-ppm group and 6 males in the 500-ppm group were not included in the

mortality or tumor analysis because they died or were taken off study after an accidental

massive dermal exposure to styrene during week 61. Body-weight gain was lower in

males in the 500- and 1,000-ppm groups and in females in the 200-, 500-, and 1,000-ppm

groups. A complete histological examination was conducted for the control and high-

exposure groups. The histologic examination for the lower-exposure groups was limited

to the target organs (nasal passages, lungs, liver, kidney, testes, and epididymides), gross

lesions, and all masses. Styrene exposure did not affect hematology, clinical chemistry,

urinalysis, or organ weights. No treatment-related effects were reported in animals

necropsied at week 52. The authors reported that there was no evidence that styrene

exposure caused significant increases of any tumor type in males or females. Treated

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female rats had decreases in pituitary adenomas and mammary adenocarcinomas

compared with controls (Table 4-8). Incidences of mammary tumors were based on the

total population rather than the number examined because these tumors are rarely found

microscopically when not seen macroscopically (Cruzan et al. 1998). There was a

positive trend for testicular tumors, but none of the pairwise comparisons was significant,

and tumor incidences were within the historical control range (0% to 13.5%). Therefore,

the differences were judged to be incidental and not treatment related.

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Table 4-8. Tumor incidences in Sprague-Dawley rats exposed to styrene by inhalation for 104 weeks

Tumor incidence (%)

Sex Exposure

conc. (ppm) Testes (interstitial

cell) Pituitary gland

(adenoma) Mammary gland

(adenocarcinoma) Male 0

50 200 500

1,000 [Trend]b

2/60 (3.3)a 2/60 (3.3) 2/60 (3.3) 4/54 (7.4)

6/52 (11.5) [P = 0.015]

31/60 (51.7) 17/60 (28.3) 28/60 (46.7) 24/54 (44.4) 20/52 (38.5)

[NS]

0/60 (0) 0/60 (0) 0/60 (0)

1/54 (1.9) 0/52 (0)

[NS] Female 0

50 200 500

1,000 [Trend]

– – – – –

45/60 (75) 42/60 (70)

35/60 (58.3) 29/60 (48.3) 31/60 (51.7) [P = 0.002N]

20/60 (33.3) 13/60 (21.7)

9/60 (15) 5/60 (8.3) 2/59 (3.4

[P = < 0.0001N] Source: Cruzan et al. 1998. conc. = concentration. aHistorical control range = 0 to 13.5% b[Cochran-Armitage exact trend test conducted by NTP. NS = not significant. A negative trend in an exposure group is indicated by N.]

4.2.3 Parenteral administration 8 Conti et al. (1988) exposed groups of Sprague-Dawley rats to styrene by either i.p. or s.c.

injection. Groups of 40 male and 40 female rats were given four i.p. injections containing

50 mg styrene in olive oil at 2-month intervals. Controls were given i.p. injections of

olive oil. Other groups of 40 male and 40 female rats received a single s.c. injection of 50

mg of styrene in olive oil. Animals were 13 weeks old at the beginning of the study and

were held until death. No treatment-related neoplasms were reported. [The studies were

markedly limited by the low and infrequent doses, short duration of styrene exposure, and

incomplete reporting.]

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4.3 Mixtures containing styrene 1

NCI (1979b) also conducted carcinogenicity studies of a mixture containing 30% β-

nitrostyrene and 70% styrene in B6C3F1 mice and F344 rats. Exposed groups included 50

animals of each sex, while control groups included 20 animals of each sex. Mice were

administered solutions in corn oil containing 87.5 or 175 mg/kg β-nitrostyrene [204 or

408 mg/kg styrene] by gavage 3 days/week for 78 weeks followed by a 14-week

observation period. Male rats were administered 150 or 300 mg/kg β-nitrostyrene [350 or

700 mg/kg styrene], and female rats were administered 75 or 150 mg/kg β-nitrostyrene

[175 or 350 mg/kg styrene] 3 days/week for 79 weeks followed by a 29-week observation

period. Control groups were gavaged with corn oil on the same schedule as the treatment

groups. All animals were approximately 6 weeks old at the beginning of the study. The

authors concluded that a sufficient number of animals survived to the end of the study in

all groups. Survival was not significantly affected by exposure in rats (both sexes) or

female mice. The probability of survival was dose-related in male mice (90% in controls,

86% in the low-dose group, and 66% in the high-dose group). Body weights were

depressed in high-dose male rats and female mice. Male mice in the low-dose group had

a significantly (P = 0.016) increased incidence (11 of 49; 22.4%) of alveolar/bronchiolar

adenoma or carcinoma compared with controls (0 of 20) (Table 4-9). The incidence in the

high-dose group was 2 of 36 (5.5%) and was not significant by pairwise comparison. No

other neoplasms in mice or rats were associated with exposure to the styrene mixture.

[However, because of poor survival of the high-dose male mice there were substantially

fewer animals at risk for late-occurring lung tumors.] The NCI concluded that “under the

conditions of this bioassay, there was no convincing evidence that a mixture of β-

nitrostyrene and styrene was carcinogenic in B6C3F1 mice or F344 rats.”

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Table 4-9. Tumor incidences in B6C3F1 mice exposed to a mixture of β-nitrostyrene and styrene for 79 weeks

Alveolar/bronchiolar tumor incidence (%)

Sex

Styrene dose

(mg/kg) Initial no.

mice Adenoma Carcinoma Combined Male 0

175 350

[Trend]b

20 50 50

0/20 a (0) 8/49 (16.3) 1/36 (2.8)

[NS]

0/20 (0) 3/49 (6.1) 1/36 (2.8)

[NS]

0/20 (0) 11/49 (22.4)*

2/36 (5.5) [NS]

Female 0 175 350

[Trend]

20 50 50

0/19 (0) 1/49 (2.0) 0/46 (0)

[NS]

0/19 (0) 1/49 (2.0) 0/46 (0)

[NS]

0/19 (0) 2/49 (4.1) 0/46 (0)

[NS] Source: NCI 1979b. NS = not significant. a (Number mice with tumor) / (number of animals examined for each tissue type). b Cochran-Armitage exact trend test conducted by NTP. NS, non-significant. * P < 0.05 (compared with controls, Fisher’s exact test).

4.4 Styrene metabolites 1

Styrene-7,8-oxide is a primary metabolite of styrene (see Section 1.3) and is listed in the

Report on Carcinogens as reasonably anticipated to be a human carcinogen based on

sufficient evidence in experimental animals (see NTP (2004) for detailed information on

the carcinogenicity of styrene oxide). IARC (1994b) also reviewed this compound and

concluded that there was “sufficient evidence in experimental animals for the

carcinogenicity of styrene-7,8-oxide.” Styrene oxide induced high incidences of both

benign and malignant tumors of the forestomach in both sexes of rats (three strains

tested) and in one strain of mice (IARC 1994b) (see Table 4-10). In addition, Lijinsky

(1986) reported liver tumors in male mice.

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Table 4-10. Summary of neoplastic lesions in mice and rats exposed to styrene-7,8-oxide by gavage

Studies

Design: dose, duration and initial

group size Comments on

study Results- male Results- female

B6C3F1 mice

Lijinsky 1986, as reviewed in IARC 1994b

0, 375, or 750 mg/kg (in corn oil) 3 days/wk, 104 wk, (97% purity)

52/sex/group

Study termination 3–4 wk after last dose

Significant increase in hepatocellular neoplasms at low dose; significant increase in forestomach tumors at both doses

Significant increase in forestomach tumors at both doses

F344 rats

Lijinsky 1986, as reviewed in IARC 1994b

0, 275 or 550 mg/kg (in corn oil) 3 days/wk, 104 wk, (97% purity)

52/sex/group

Study termination 3–4 wk after last dose



Sprague-Dawley rats

Conti et al. 1988, Maltoni et al. 1979, as reviewed in IARC 1994b

0, 50, 250 mg/kg (in olive oil) 4–5 d/wk, 52 wks (purity not specified)

40/sex/group

Rats held after dosing until death

Significant dose- dependent increase in forestomach tumors

Significant dose- dependent increase in forestomach tumors

BD IV inbred rats

Ponomarkov et al. 1984, as reviewed in IARC 1994b

200 mg/kg (in olive oil) 14 dams dosed on prenatal day 17; progeny dosed once per wk at 100–150 mg/kg, 96 wk starting at 4 wk of age (99% purity)

62 females and 43 males

Dams of control progeny were not dosed; control progeny of 55 females and 49 males dosed with vehicle; study terminated at 120 wk

Significant increase in forestomach tumors

Significant increase in forestomach tumors

4.5 Summary 1

The carcinogenicity of styrene has been investigated in rats and mice by several routes of

exposure and the results are summarized in Tables 4-11 and 4-12. [Many of the studies

were severely limited in their ability to detect carcinogenic effects because of inadequate

study design (low doses, short treatment or short study duration, small group size) or

intercurrent disease and high mortality (e.g., pneumonia), or the studies were

inconclusive because of limited reporting (tumor diagnosis, statistical methodology).]

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In mice, gavage studies in both sexes for three strains, an inhalation study in both sexes

of one strain, and one i.p. study in females were found in the literature and reviewed.

[The oral gavage studies in B6C3F1 mice (NCI 1979a) and the inhalation studies in CD-1

mice (Cruzan et al. 2001) were the most informative of the carcinogenicity studies.]

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Male B6C3F1 mice had a statistically significant dose-response trend for

alveolar/bronchiolar adenoma and carcinoma (combined) that was supported by a

significantly increased incidence of these lung tumors in the high-dose group. The

authors questioned the significance of these lung tumors because the incidence in the

control group was unusually low compared with historical untreated controls. However,

the concurrent vehicle controls were within the range of historical vehicle controls from

the same source, same study protocol, and same chronological window. Further, the

tumor incidence in the high-dose group was significantly increased compared with these

historical vehicle controls. A dose-related trend in female B6C3F1 mice was also

observed for hepatocellular adenoma. Significantly increased incidences of

alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma

(combined) were also observed in male and female CD-1 mice exposed to styrene by

inhalation. In each sex, three treatment groups (males, 40, 80, 160 ppm; females, 20, 40,

160 ppm) showed increases in these tumors. The high-dose (160-ppm) female mice had

an increased incidence of alveolar/bronchiolar carcinoma. A significant trend in

hepatocellular adenoma in female mice was also reported, but the pairwise comparison

between treated and control animals was not significant.

In a short-term oral gavage study in O20 mice, a strain with a high spontaneous incidence

of lung tumors, significantly higher incidences of lung tumors (adenoma and carcinoma

combined) were observed in both males and females compared with vehicle controls.

In rats, gavage studies in three strains, and three inhalation studies in one strain, and a

drinking-water study in one strain were reviewed. The oral gavage studies in F344 rats

(NCI 1979a) and the inhalation studies in Sprague-Dawley rats (Cruzan et al. 1998) were

the most informative of the carcinogenicity studies. Neither study showed an increase in

tumor incidences in styrene-treated rats, although Sprague-Dawley rats exhibited a dose-


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related reduction in pituitary and mammary gland tumors. A significant trend in

interstitial testicular tumors was found in rats, but the pairwise comparison between

treated and control animals was not significant. In the inhalation study reported by Conti

et al. (1988), there was a dose-related increase in the incidences of malignant mammary

gland tumors; treatment-related and statistically significant incidences of these tumors

were seen in the top three dose groups. The drinking-water study in Sprague-Dawley rats

did not report any dose-related carcinogenic effects; however, statistical reanalyses of

study data indicated a marginal increase in mammary fibroadenoma in high-dose female

rats and a significant dose-related trend. For the unpublished inhalation study by Jersey et

al. (1978), a statistically significant increase in mammary adenocarcinoma in the low-

dose, but not high-dose group was reported in several reviews of this study. [There was

an inconsistent association of mammary-gland tumors and styrene treatment across these

studies]. Elevated leukemia/lymphosarcoma were observed in both treatment-related

groups of female Sprague-Dawley rats in one inhalation study (Jersey et al. 1978).

[However the study was limited by lack of information on whether the leukemia was

lymphocytic in nature.]

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No increase in alveolar/bronchiolar tumor incidence was observed in female rats exposed

to a mixture of 70% styrene and 30% β-nitrostyrene. An increase in lung tumors (low-

dose group only) was observed in male mice exposed to this styrene/β-nitrostyrene

mixture. [Substantial mortality in the high-dose group could have precluded the

observation of late-occurring tumors, such as the lung, in many animals.]

Uncertain findings include hepatocellular adenomas in female mice (NCI 1979a) and

interstitial testicular tumors in rats (Cruzan et al. 1998), both of which were statistically

significant by trend but not by pairwise comparison between treated and control animals.


Table 4-11. Summary of studies in mice

Results

Studies

Design: dose, duration and

initial group size Limitations of

study Males Females Oral B6C3F1 mice NCI 1979a

150 or 300 mg/kg (in corn oil by gavage), 5 days/wk by gavage, 78 wk Controls – 20/sex Treated – 50/dose level/sex

Limited control group size

Significant increase and dose-related trend in lung adenoma and carcinoma combined

Dose-related increase in hepatocellular adenoma

O20 mice Ponomarkov and Tomatis 1978

1,350 mg/kg (in olive oil by gavage) once on prenatal day 17 & weekly postweaning for 16 wk. Controls – 20 males, 22 females Dosed – 45 males, 39 females

High mortality in treated animals; only one treatment group; short dosing duration; small control groups

Significant increase in lung tumors

Significant increase in lung tumors

C57Bl mice Ponomarkov and Tomatis 1978

300 mg/kg (in olive oil by gavage) once prenatal day 17 and weekly postweaning until death. Controls – 12 males, 13 females Dosed – 27 males, 27 females

Only one treatment group; low dose; limited reporting; small group size, particularly controls

No significant increase in tumors

No significant increases in tumors

Inhalation CD-1 mice Cruzan et al. 2001

20, 40, 80 or 160 ppm, 6 h/d, 5 d/wk, 98–104 wk 50 animals/group/sex

No major limitations

Significant increase and dose-related trend in lung adenoma and combined adenoma and carcinoma

Significant increase and dose-related trend in lung adenoma, carcinoma, and combined adenoma and carcinoma

Intraperitoneal Female A/J mice Brunnemann et al. 1992

Total 100 mg/kg in divided doses, 3/wk, held for 20 wk after last injection 25 animals/group

Only one treatment group, limited reporting, small group size

NA No significant increase in lung tumors

NA = not applicable.

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Table 4-12. Summary of studies in ratsa

Studies


group size Limitations of

study Results male Results female Oral F344 rats

NCI 1979a

500, 1000, or 2000 mg/kg (in corn oil, by gavage), 5 d/wk, 78 wk (mid- & high dose) or 103 wk (low dose)

Controls – 20/sex

Treated – 50/ dose group/sex

Poor survival of high dose groups, small control group



BD IV rat

Ponomarkov and Tomatis 1978

1,350 mg/kg (in olive oil by gavage) once prenatal day 17 & 500 mg/kg weekly postweaning for lifespan

Controls – 39 females/36 males

Dosed – 71 females/73 males

Limited dosage regimen, once/wk dosing, limited reporting



Sprague-Dawley rats

Conti et al. 1988

50 or 250 mg/kg, 4–5 d/wk (in olive oil by gavage) for 52 wk and held until death

40/dose group/sex

Mortality in high-dose females, short treatment duration, low doses, limited reporting



Sprague-Dawley rats

Beliles et al. 1985

125 or 250 ppm in drinking water (7.7–14 mg/kg/d in males (50/group) and 12–20.5 mg/kg/d in females (70/group) for 104 wk; controls 104 females and 76 males

Low doses, limited reporting


Small increase in mammary fibroadenoma

Inhalation Sprague-Dawley rats

Conti et al. 1988

25, 50, 100, 200 or 300 ppm (99.8% purity), 4 h/d, 5 d/wk for 52 wk and held until death

Controls – 60/sex Treated – 30/sex/dosegroup

Limited dosing regimen, limited reporting


malignant mammary tumors increased in all groups, with significant trend

Sprague-Dawley rats

600 or 1,200/1,000 ppm, 6 h/d, 5 d/wk, for 18.3 mo (males) or 20.7

Original report and data not available in


Small increase in leukemia/lymphosarcoma, with a

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Studies


group size Limitations of

study Results male Results female Jersey et al. 1978, as cited cited in Huff 1984, WHO 1983, McConnell and Swenberg 1993, 1994, Cohen et al. 2002.

mo (females)

96/sex/treatment group

published literature; limited reporting in reviews, high incidence of pneumonia

significant trend

Increase in malignant mammary tumors in low-dose group

Sprague-Dawley rats

Cruzan et al. 1998

0, 50, 200, 500 or 1000 ppm, 6 h/d, 5 d/wk for 104 wk

70/sex/group

No major limitations

Positive dose- related trend in benign interstitial testicular tumors (incidence within historical control range 0-13.5%)

Pituitary and malignant mammary tumors decreased in all dose groups (negative trend)

aThe parenteral administration (i.p. or s.c.) study by Conti is not presented here because of the limitations of low and infrequent doses, short duration of exposure, and incomplete reporting.



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5 Other Relevant Data 1

This section discusses relevant mechanistic data and other information needed to

understand the toxicity and potential carcinogenicity of styrene. It includes information

for styrene on absorption, distribution, metabolism, and excretion (Section 5.1), toxicity

(Section 5.2), interspecies differences in metabolism, toxicity, and toxicokinetics (Section

5.3), genetic and related effects (Section 5.4), and mechanistic studies and considerations

(Section 5.5). A summary is provided in Section 5.6.

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5.1 Absorption, distribution, metabolism, and excretion 8

This section describes absorption (5.1.1), distribution (5.1.2), metabolism (5.1.3), and

excretion (5.1.4) of styrene in humans and experimental animals.

5.1.1 Absorption 11 Studies in humans and experimental animals show that styrene is absorbed following

inhalation, ingestion, or skin contact. Human data are presented in Section 5.1.1.1 and

experimental animal data are presented in Section 5.1.1.2.

5.1.1.1 Humans 15 Styrene may be absorbed following inhalation, ingestion, or skin contact; however, the

predominant route in occupational settings is inhalation (ATSDR 1992, IARC 1994a,

2002). Food is also an important source of exposure for the general population (see

Section 2.3.4 and 2.4). In humans, approximately 60% to 70% of inhaled styrene is

absorbed. No data were identified regarding oral absorption of styrene in humans, but

several studies were available that evaluated dermal absorption. Dutkiewicz and Tyras

(1968) reported that the rates of absorption of liquid styrene through the skin of the hand

and forearm of a man were 9 to 15 mg/cm2/h. When applied as an aqueous solution at

concentrations of 66.5 to 269 mg/L, the rates of absorption were 0.040 to 0.18 mg/cm2/h.

Dermal absorption of residual styrene monomer from polystyrene-containing personal

care products was demonstrated using in vitro diffusion-cell techniques (Kraeling and

Bronaugh 2005). When 14C-styrene (4.1 μg/cm2) was applied to human skin as an oil-in-

water emulsion that simulated cosmetic products, only 1.3% of the applied styrene was

absorbed (1.2% absorbed into the receptor fluid and 0.1% remaining in the skin after 24


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hours). Although absorption was low, it was rapid with peak absorption occurring at

about 6 hours. The total recovery of styrene in this study was only 1.5%. The low

recovery was attributed to volatilization of styrene from the emulsion. The dermal

absorption rate of styrene in human volunteers who dipped one hand into liquid styrene

for 10 to 30 minutes was low (about 1 μg/cm2/min) (Berode et al. 1985). In another

study, dermal absorption was determined by measuring styrene and styrene metabolites in

blood, exhaled air, and urine in 10 volunteers who were exposed to styrene vapors (with

respiratory protection) at 600 ppm for 3.5 hours (Riihimäki and Pfäffli 1978). Dermal

absorption of styrene vapors was estimated to be about 0.1% to 2% of the estimated

exposure from inhalation. In a similar experiment, Wieczorek (1985) measured styrene

metabolites in the urine of four volunteers exposed to styrene vapor at 1,300 to 3,200

mg/m3 [300 to 740 ppm] for 2 hours and estimated that dermal absorption was about 5%

of the amount absorbed via the respiratory tract. Limasset et al. (1999) compared urinary

excretion of styrene metabolites in four groups of workers in the fiberglass-reinforced

polyester (reinforced plastics, see Section 2.5.1) industry. The groups performed the same

task at the same time and place but wore different types of protective equipment (total

body protection, skin protection only, respiratory protection only, or no protection).

There was no significant difference in urinary excretion of styrene metabolites in the

group with total protection compared with the group using respiratory protection only.

The authors concluded that percutaneous absorption of styrene vapor did not make an

important contribution to the body burden of styrene-exposed workers. However, Luderer

et al. (2005) estimated that in situations of prolonged and repeated contact with liquid

styrene, dermal uptake could be equivalent to inhalation exposure at the lower range of

occupational styrene concentrations (1 to 2 ppm).

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5.1.1.2 Experimental animals 25 Styrene was absorbed in laboratory rodents exposed to styrene vapors or by oral

administration, intraperitoneal injection, and skin application (ATSDR 1992, IARC

1994a, 2002). Inhalation studies in rats at concentrations ranging from 50 to 2,000 ppm

for 5 hours or 80 to 1,200 ppm for 6 hours indicated rapid uptake with styrene

concentrations in blood reaching maximal values at the end of the exposure period. In

one study, a 15-fold increase in exposure concentration (80 to 1,200 ppm for 6 hours)


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resulted in a 63-fold increase in blood levels and indicated saturation of styrene

metabolism at high concentrations. Morris (2000) examined the uptake of styrene in

surgically isolated upper respiratory tracts of Sprague-Dawley rats and CD-1 mice. The

average uptake efficiency in rats ranged from 24% with exposure to styrene at 5 ppm to

about 9% or 10% with exposure at 100 or 200 ppm. The steady-state uptake decreased

with increasing concentration. In mice exposed to the same range of styrene

concentrations, the average uptake efficiency ranged from 42% (5 ppm) to 10% (200

ppm); however, uptake efficiency did not maintain a steady state, but declined steadily

during exposure. Pretreatment with the cytochrome P450 (CYP450) inhibitor metyrapone

significantly reduced uptake efficiency in both rats and mice and abolished the

concentration dependence. The loss of concentration dependence and the observation that

metyrapone pretreatment also caused uptake efficiency to achieve steady state in mice led

the author to conclude that both the concentration dependence and the non-steady–state

behavior in mice likely were due to styrene metabolism.

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In a study of dermal absorption of styrene vapor in male F344 rats, the maximum blood

concentration of about 10 μg/mL was achieved after 4 hours of exposure to 3,000 ppm

(McDougal et al. 1990). The skin permeability constant was 1.75 cm/h. When exposure

was by both inhalation and skin absorption, styrene uptake via skin exposure was

estimated to be 9.4% of the total absorbed. In another study of dermal absorption in F344

rats, the peak blood concentration was 5.3 μg/mL when 2 mL of neat [undiluted] styrene

was administered in a sealed dermal cell; absorption was less when the styrene was

diluted with water (Morgan et al. 1991). Sandell et al. (1978) exposed adult male Wistar

rats to cutaneous doses (0.5 or 3.0 g/kg) of styrene daily for 7 consecutive days. They

reported that rat skin was easily penetrated by styrene as evidenced by changes in

detoxifying enzyme activity in the liver but not in the lung.

5.1.2 Distribution 26 This section discusses distribution of styrene and its metabolites in humans (5.1.2.1) and

rodents (5.1.2.2). Absorbed styrene is widely distributed from the blood to other body

tissues (ATSDR 1992).


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5.1.2.1 Humans 1 The observation that partition coefficients for styrene between body tissues and air are

high (4,100 for fat, 84 to 154 for other organs, and 59 for blood) led to the suggestion that

styrene would accumulate in subcutaneous fat (IARC 1994a, 2002). However, in a study

of styrene-exposed workers, urinary excretion of mandelic acid and phenylglyoxylic acid

did not increase during a work week, leading the authors to conclude that styrene did not

accumulate (Pekari et al. 1993). IARC (2002) noted that pharmacokinetic analysis of the

disposition of styrene does not indicate that styrene accumulates in subcutaneous fat.

Ramsey et al. (1980) exposed four healthy volunteers to 80-ppm styrene for 6 hours and

concluded that styrene would not accumulate in the human body. The estimated half-life

of styrene in subcutaneous fat in humans is between 2 and 5 days (ATSDR 1992, IARC

1994a).

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5.1.2.2 Experimental animals 13 In a study of tissue distribution of styrene and its metabolites in mice exposed to styrene

via i.p. injection, the highest concentrations of unchanged styrene were detected in fat,

pancreas, liver, and brain. However, polar metabolites were detected in the liver, kidneys,

lungs, and plasma only (Löf et al. 1983). In rats orally exposed to styrene, Plotnick and

Weigel (1979) found the highest concentrations of styrene in the kidney, liver, and

pancreas.

In one study reviewed by IARC (1994a), the concentration of styrene in the blood of

male Wistar rats exposed to styrene vapor at 50 to 2,000 ppm, or injected with styrene

intravenously (i.v.) at doses of 1.3 to 9.4 mg/kg b.w., indicated saturation of metabolic

elimination at higher concentrations. The apparent volume of distribution, however, was

not dependent on exposure level and was approximately 10 times the blood volume of the

animals, indicating that styrene distributed extensively to the tissues. The concentration

of styrene in perirenal fat was about 10 times the concentration seen in any other organ.

The reported biological half-life of styrene in rats [strain not specified] was 6.3 hours,

and the half-lives in blood, liver, kidney, spleen, muscle, and brain were between 2.0

hours and 2.4 hours.


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Boogaard et al. (2000a) exposed rats and mice to [ring-U-14C]styrene by nose-only

inhalation and performed quantitative whole-body autoradiography on sections taken

from one rat and two mice. Radioactivity was detectable in over 40 different tissues, but

its concentration in most tissues was lower than in blood. Tissues where the concentration

was higher than in blood included the liver and kidney cortex in both rats and mice, with

higher levels in mice for both tissues. Radioactivity levels were clearly higher in the

lungs than in the blood of mice, but were higher in the blood than in the lungs of rats. The

radioactivity in the lungs of mice was located in discrete regions that the authors equated

with the bronchi. In both species, the nasal mucosa contained higher levels of

radioactivity than the blood (> 3 times as much in rats and 2 to 13 times as much in

mice), with most of it residing in the olfactory mucosa rather than the respiratory mucosa.

The authors also noted that their measurements of radioactivity in fat indicated that

styrene was stored in fat during exposure, but was released rapidly from fat after the

exposure period ended. The high levels of radioactivity in the kidney also were transient

and were most likely related to clearance of radiolabeled styrene through the kidney.

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5.1.3 Metabolism 16 This section describes the metabolic pathways for styrene in humans (5.1.3.1) and

experimental animals (5.1.3.2), differences in styrene metabolism among tissue and cell

types (5.1.3.3), metabolic enzyme activity in human lung in general (5.1.3.4), the roles of

specific metabolic enzymes in biotransformation of styrene (5.1.3.5), and detoxification

of styrene metabolites (5.1.3.6).

5.1.3.1 Humans 22 The primary and secondary metabolic pathways for styrene in humans are shown in

Figure 5-1. The available data indicate that styrene metabolism becomes saturated at air

concentrations greater than 200 ppm in humans, rats, and mice (ATSDR 1992).


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Figure 5-1. Styrene metabolism in humans Source: Manini et al. 2002b.

Bold arrows show the main pathway. PHEMAs are four diastereoisomers: (R,R)- and (S,R)-N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine and (R,R)- and (S,R)-N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine. Abbreviations: CYP2E1 and CYP2B6 = cytochrome P450 monooxygenase, mEH = microsomal epoxide hydrolase, ADH = alcohol dehydrogenase, AlO = aldehyde oxidase, ALDH = aldehyde dehydrogenase, XO = xanthine oxidase, DC = decarboxylase, GSH = glutathione, GSTs = glutathione S-transferases, γ-GT = gammaglutamyl transpeptidase, NAcT = N-acetyltransferase.

The main route of styrene metabolism in humans produces the terminal metabolites

mandelic and phenylglyoxylic acids by way of the intermediate styrene-7,8-oxide, which

is subsequently hydrolyzed to styrene glycol (phenylethylene glycol) (IARC 1994a,

Sumner and Fennell 1994). Styrene-7,8-oxide contains a chiral carbon and can exist as

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either the R- or S-enantiomer. More than 90% of the styrene retained in humans is

initially activated to genotoxic styrene-7,8-oxide, with subsequent conversion to

detoxification products. Carlson et al. (2000) detected the metabolism of styrene to

styrene-7,8-oxide in 6 of 6 human liver microsomal preparations and 1 of 6 lung

microsomal preparations collected from 12 individuals during surgical procedures or at

autopsy. Liver microsomes showed a much higher metabolic activity than lung

microsomes.

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Korn et al. (1994) reported a linear correlation between the concentrations of styrene-7,8-

oxide in the blood of workers and the concentration of styrene in air. The steady-state

level of styrene-7,8-oxide was about 1 μg/L at styrene concentrations of 20 ppm.

Johanson et al. (2000) exposed 4 healthy male volunteers to 50-ppm styrene for 2 hours

during light physical activity. Maximum concentrations of styrene-7,8-oxide in blood

ranged from 2.5 to 12.2 nM and were observed in the first samples collected shortly after

exposure had stopped. No styrene-7,8-oxide was detected in blood samples collected 23.5

hours after exposure. Minor styrene metabolites identified in humans include mercapturic

acid derivatives of styrene-7,8-oxide (Maestri et al. 1997) (which arise from glutathione

conjugation of styrene-7,8-oxide), 4-vinylphenol (4-hydroxystyrene) (Pfäffli et al. 1981),

1-phenylethanol (Korn et al. 1985), 2-phenylethanol (Korn et al. 1985), and glucuronic

acid and sulfur conjugates of hydroxylated styrene metabolites (Manini et al. 2002b).

Formation of 4-vinylphenol indirectly indicates intermediate formation of the 3,4-arene

oxide; formation of 2-vinylphenol (not shown in Figure 5-1), indirectly indicates

intermediate formation of styrene-2,3-oxide. Urinary 4-vinylphenol sulfates and

glucuronates have been identified in volunteers and occupationally exposed workers, and

this metabolic pathway was shown to account for approximately 0.5% to 1% of the total

excretion of styrene metabolites (Manini et al. 2003).

5.1.3.2 Experimental animals 26 Metabolism of styrene in various animal species has been reviewed by IARC (1994a,

2002) and by Sumner and Fennel (1994). As in humans, the first step in metabolism is

usually the epoxidation of styrene to styrene-7,8-oxide in a NADPH-dependent reaction

catalyzed by CYP enzymes (Figure 5-1). Styrene-7,8-oxide is further metabolized to


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styrene glycol by epoxide hydrolase or conjugated with glutathione to form mercapturic

acid metabolites. 1-Phenylethanol and 2-phenylethanol also have been identified as

urinary metabolites in rats. The liver has the highest activity for formation of styrene-7,8-

oxide and its subsequent conversion to styrene glycol. These metabolic steps also occur

in lung and kidney, but not in heart, spleen, or brain. This preferential metabolism of

styrene in the liver was found consistently in all species examined (male and female

Sprague-Dawley rats, CD-1 mice, New Zealand rabbits, and Dunkin-Hartley guinea-

pigs).

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A second metabolic pathway results in formation of 4-vinylphenol, which is produced in

very small amounts in rats (0.1% of styrene dose) (Bakke and Scheline 1970) and

humans (Manini et al. 2003); this pathway is postulated to involve styrene-3,4-oxide as

an intermediate (Pantarotto et al. 1978). No 4-vinylphenol was detected in in vitro

experiments with mouse and rat lung and liver microsomal preparations incubated with

styrene, but the authors suggested that rapid metabolism of 4-vinylphenol might explain

their failure to detect the metabolite (Carlson et al. 2001). When the metabolism of

4-vinylphenol was tested in the same system, the metabolic rate was 3 times as high in

mouse liver microsomes as in rat liver microsomes and 8 times as high in mouse lung

microsomes as in rat lung microsomes. Boogaard et al. (2000a) reported that the

percentage of 14CO2 derived from ring-labeled styrene was 3 to 4 times as high in mice as

in rats and suggested that this might indicate formation of reactive ring-opened

metabolites in mouse lung, which would likely involve ring oxidation, as postulated for

the formation of 4-vinylphenol. Differences in styrene metabolites formed by ring

oxidation have been proposed as a possible explanation for the interspecies differences in

susceptibility to lung tumors in experimental animals (see Sections 5.1.3.5, 5.2.2.2, and

5.5.3).

5.1.3.3 Tissue type, lung cell types, and metabolism 26 Green et al. (2001b) examined metabolism of styrene to styrene-7,8-oxide and

detoxification of styrene-7,8-oxide in vitro by nasal epithelium of mice, rats, and humans.

The rates of styrene metabolism to styrene-7,8-oxide were higher in rat and mouse nasal

tissues, both olfactory and respiratory, than in liver. No metabolism of styrene to styrene-


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7,8-oxide was detected in 9 samples of human nasal epithelium (8 S9 fractions and 1

microsomal fraction) at a limit of detection of 0.04 nmol/min per mg of protein.

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A significant proportion of the oxidative metabolizing capacity of the rodent lung occurs

in Clara cells in mice and type II cells in rats (Pinkerton et al. 1997). Clara cells are the

target cells for styrene-induced pneumotoxicity (Cruzan et al. 1997). Hynes et al. (1999)

investigated the roles of Clara cells and type II cells in styrene metabolism in rats and

mice. Enriched Clara-cell and type II alveolar-cell fractions were obtained from lungs of

male CD-1 mice and male Sprague-Dawley rats. The mouse and rat cell preparations

metabolized styrene to R- and S-styrene-7,8-oxides; however, the R/S ratio was higher in

mice than in rats. The metabolizing activity of mouse Clara cells was several-fold higher

than that of rat Clara cells (Table 5-1). Metabolism was higher in fractions enriched for

Clara cells compared with fractions enriched for type II cells. When the activities for the

two fractions were solved as simultaneous equations (considering the percentage

enrichment of each fraction), practically all the metabolizing activity was attributed to

Clara cells.

Table 5-1. Production of R- and S-enantiomers of styrene-7,8-oxide by cell preparations enriched in either Clara cells or type II cells from rat and mouse lungsa

Production (pmol/106 cells per min)b

% Clara cells % Type II cells R-enantiomer S-enantiomer R/S ratio

Male CD-1 mouse (4 experiments) 18.3 ± 3.5 33.5 ± 4.9 19.4 ± 4.1 6.9 ± 2.2 3.62 ± 1.09 55.8 ±8.0 6.5 ± 2.5 83.3 ± 27.7 23.0 ± 8.2 3.98 ± 0.75 Male Sprague-Dawley rat (3 experiments) 12.8 ± 3.2 42.3 ± 4.1 3.7 ± 1.1 8.0 ± 2.6 0.47 ± 0.01 37.3 ± 9.0 4.0 ± 1.0 11.2 ± 3.6 11.0 ± 3.2 1.02 ± 0.09 Source: Hynes et al. 1999. aAll values are mean ± SE. bCalculated on the basis of total number of nucleated cells.

Boogaard et al. (2000b) compared DNA adduct formation in rat and mouse liver and

lung, and in fractions enriched in type II and Clara cells isolated from rat and mouse lung

(see Section 5.4.3.1). DNA adduct profiles in liver and lung tissue were similar, but the

adduct levels were significantly lower in lung. However, DNA adduct profiles in mice

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and rats showed both quantitative and qualitative differences. These differences suggest

that different reactive metabolites are formed in rats and mice. Clara cells are the

predominant cell type in mouse lung, while type II cells predominate in rat lung.

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Human lung also contains Clara cells (primarily in the bronchiolar epithelium) but the

morphology is different from that seen in rodents. Pinkerton et al. (1997) reported that

the most striking difference was the low proportion of agranular endoplasmic reticulum

in human nonciliated bronchiolar epithelial cells (3.1% of the cellular components)

compared with 55% in the mouse and 66% in the rat. Human terminal airways do not

have significant numbers of Clara cells; however, the contribution of Clara cells to the

proliferation compartment of normal human tracheobronchial epithelium is substantial,

demonstrating a role of the Clara cell in the maintenance of the normal epithelium of the

distal conducting airways in humans. This concept was demonstrated in the study by

Boers et al. (1999). These authors evaluated the number of Clara cells from normal tissue

taken from seven lungs obtained by autopsy. The number of Clara cells in the terminal

and respiratory bronchioles were 11 ± 3% and 22 ± 5%, respectively. The proximal

airway epithelium (bronchi and bronchioles) was virtually devoid of Clara cells. The

overall proliferation compartment of the conducting airway epithelium was 0.83 ±

0.47%; the contribution of Clara cells was 9%. In the terminal bronchioles 15% of

proliferating airway epithelial cells were Clara cells, and in the respiratory bronchioles

this percentage increased to 44%.

5.1.3.4 Metabolic enzyme activity in human lung 21 The ability of lung cells to metabolize styrene to potentially tumorigenic molecules could

be an important mechanistic factor in explaining the differences in the formation of lung

tumors in experimental animals, particularly the development of lung tumors in mice but

not in rats exposed to styrene. To understand the relevance of these findings to humans, it

is important to examine the potential for human lung cells to metabolize styrene to the

molecules identified as potential tumorigenic intermediates in animal studies, and that

metabolism will depend on the expression cytochrome P450 isozymes. This section

discusses that expression.


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Human lung has been reported to contain either mRNA or protein for the following P450

isozymes: CYP1A1, CYP1A2, CYP2A6, CYP2A13, CYP1B1, CYP2B6, CYP2C8,

CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2F1, CYP2J2, CYP2S1, CYP3A4,

CYP3A5, CYP4B1, CYP5A1, CYP7B1, CYP8A1, CYP27, and CYP51 (Ding and

Kaminsky 2003, Hukkanen et al. 2002, Karlgren et al. 2005, Nishimura et al. 2003,

Pelkonen and Raunio 1997, Seliskar and Rozman 2007, Somers et al. 2007, Zhang et al.

2006). Although levels of most P450 enzymes are reported to be lower in lung compared

with liver (Somers et al. 2007), CYP2A13, CYP2F1, CYP2S1, CYP3A5, and CYP4B1

are preferentially expressed in the lung (Ding and Kaminsky 2003, Thum et al. 2006).

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Xenobiotic metabolism in human lung occurs primarily in bronchial epithelial cells, Clara

cells, type II pneumocytes, and alveolar macrophages; while in rodents and rabbits,

metabolism is highest in Clara cells and type II pneumocytes. The CYP2F1 isoform of

cytochrome P450 is the human homolog of the Cyp2f2 isoform expressed in mouse lung

(see below) and the CYP2F4 isoform expressed in rat lung (Baldwin et al. 2005). The

presence of a cDNA for CYP2F1 in a human lung library was first reported by Nhamburo

et al. (1990). The mRNA for CYP2F1 has been shown by RT-PCR amplification to be

present in human lung tissue and broncho-alveolar macrophages (Raunio et al. 1999) and

in human bronchial biopsy and trachea and lung tissue (Bieche et al. 2007, Thum et al.

2006).

Sheets et al. (2004) reported that A549 human alveolar epithelial type II

(adenocarcinoma) lung cells were capable of metabolizing benzene, and the activity

decreased significantly (51%; P < 0.05) in the presence of 5-phenyl-1-pentyne (5P1P), a

P450 inhibitor. 5-Phenyl-1-pentyne is an effective inactivator of CYP2E1 as well as

CYP2F2 and CYP2F1 (Roberts et al. 1998, Simmonds et al. 2004). The authors

concluded that CYP2F1 was important in benzene metabolism in this human lung cell

line. BEAS-2B cells overexpressing CYP2F1 also were reported to have a significant (P

< 0.05) increase in cytotoxicity resulting from bioactivation of 3-methylindole to 3-

methyleneindolenine (Nichols et al. 2003).


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5.1.3.5 Metabolic enzyme studies 1 Most styrene metabolism is mediated enzymatically, but nonenzymatic epoxidation of

styrene has been demonstrated in human erythrocytes in vitro (Tursi et al. 1983). These

experiments showed a linear relationship between styrene oxidation and the molar

fraction of oxyhemoglobin, indicating that oxyhemoglobin rather than free oxygen

radicals are involved in the reaction. The enzymatic metabolism of styrene, and the

contributions of various cytochromes P450 in animal tissues have been studied through

the use of chemical inhibitors and antibodies to specific cytochromes (IARC 2002), and

Cyp2E1 knockout mice (Carlson 2003, 2004a). These studies show that there are tissue

differences in the enzymes responsible for styrene oxidation. A large number of human

liver and lung CYP isoenzymes are capable of oxidizing styrene at the 7,8-position, but

the most important appear to be CYP1A2, CYP2B6, CYP2E1, CYP2F1, CYP2C8, and

CYP3A4. The enzymes involved in the formation and detoxification of styrene

metabolites in humans and experimental animals are discussed in this section.

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The biotransformation of styrene may be affected by inducers or inhibitors of xenobiotic

metabolism. Metabolism of styrene in vivo in female Wistar rats and in vitro in liver

microsome preparations from male Wistar rats was increased by administration of

sodium pentobarbital (IARC 1994a). Co-exposure to acetone increased urinary styrene

metabolites in male Han/Wistar rats, and i.p. injection of toluene suppressed styrene

metabolism in Wistar rats. In a perfused rat liver system, co-administration of ethanol

decreased the uptake and metabolism of styrene.

Nakajima et al. (1994a) compared the rate of formation of styrene glycol from styrene in

human, rat, and mouse liver microsomes, and in human lung microsomes. At low styrene

concentrations (0.085 mM), the rate was highest in mouse liver microsomes (2.43 ± 0.29

nmol/(mg of protein·min)) and lowest in humans (0.73 ± 0.45 nmol/(mg of protein·min));

however, at a higher styrene concentrations (1.85 mM), the rate was highest in the rat

(4.21 ± 0.72 nmol/(mg of protein·min)) but remained lowest in humans (1.91 ± 0.84

nmol/(mg of protein·min)). The rate of styrene glycol formation in human lung was less

than 1% of that in human liver. The specific P450 forms responsible for the metabolic

activity were determined by analyzing cDNA-expressed individual P450 forms produced


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in cultured hepatoma G2 cells by recombinant vaccinia viruses. Of the 12 human P450

forms studied, 9 were able to catalyze styrene oxidation. All the P450 forms studied,

except CYP2F1 and CYP4B1, are expressed in the liver. CYP2B6 (119.6 nmol/(dish·2h))

and CYP2E1 (63.4 nmol/(dish·2h)) were the most active forms in human liver

microsomes, while CYP2F1 (103.9 nmol/(dish·2h)) was most active in human lung

microsomes. CYP1A2 (63.2 nmol/(dish·2h)) and CYP2C8 (47.6 nmol/(dish·2h)) showed

intermediate activity. Less active forms included CYP3A3, CYP3A4, CYP3A5, and

CYP4B1 (~12 to 24 nmol/(dish·2h)), while little detectable activity was reported for

CYP2A6, CYP2C9, and CYP2D6. Mouse Cyp1a2 (85.0 nmol/(dish·2h)) was more active

than mouse Cyp1a2 (17.2 nmol/(dish·2h)). Mouse Cyp1a2 and human CYP1A2 are

orthologous counterparts, but the human form had about 3.5-fold greater activity than the

mouse form. Rat CYP2B1 was the most active P450 investigated (198.8 nmol/(dish·2h))

and had more than twice the catalytic activity of rat CYP2B2 even though they have

similar amino acid sequence homology.

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Fukami et al. (2008) reported that CYP2A13, a human cytochrome P450 expressed

predominantly in the respiratory tract, had the highest catalytic activity for the formation

of styrene-7,8-oxide from styrene when compared with CYP2A6 and CYP2E1. These

enzymes have overlapping substrate selectivity. The CLint values calculated from the

initial slope of velocity plotted against the substrate concentration values were 46.8 for

2A13, 17.2 for 2A6, and 18.5 for 2E1.

The effects of CYP-specific inhibitors on formation of R- and S-enantiomers of styrene-

7,8-oxide were investigated by Hynes et al. (1999) through the use of enriched Clara cell

and type II alveolar cell fractions from lungs of male CD-1 mice and male Sprague-

Dawley rats. Cyp2e1 and Cyp2f2 were found to be the most important isoforms. Hynes et

al. reported that the mouse Clara cell preparation metabolized styrene to both

enantiomers. CYP-specific inhibitors (diethyldithiocarbamate for Cyp2e1, 5-phenyl-1-

pentyne for Cyp2f2, α-naphthoflavone for Cyp1a, and α-

methylbenzylaminobenzotriazole for Cyp2b) were used to identify the cytochromes

responsible. The Cyp1a inhibitor did not inhibit styrene-7,8-oxide formation, and the

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the R- and S-enantiomers by approximately 34%, similar to the effect of SKF-525A, a

nonspecific CYP inhibitor. In a previous study, Carlson (1997b) reported that

diethyldithiocarbamate inhibited styrene metabolism in lung microsomal preparations by

more than 50%. In contrast, Hynes et al. did not find a significant effect of this Cyp2e1

inhibitor on styrene metabolism in the Clara cell preparation and concluded from the

results of both microsomal and isolated cell studies that Cyp2e1 and Cyp2f2 were the

primary cytochromes responsible for styrene metabolism in the lung.

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In another study, styrene metabolism in rat liver microsomes was decreased by antibodies

to CYP2C11/6, CYP2B1/2, CYP1A1/2, and CYP2E1, with the strongest effect for anti-

CYP2C11/6 (Nakajima et al. 1994b). CYP2C11/6, CYP2B1/2, and CYP1A1/2 were

more important at high substrate concentrations, while CYP2E1 contributed more at low

substrate concentrations. Metabolism by lung microsomes was inhibited only by anti-

CYP2B1/2.

Kim et al. (1997) used antibodies against specific human CYP isoenzymes and compared

rates of styrene glycol formation by microsomes isolated from human livers. They

identified CYP2E1 and CYP2C8 as the most important metabolic enzymes at a low

styrene concentration (0.085 mM) and CYP2B6 and CYP2C8 as most prominent at a

high styrene concentration (1.8 mM). CYP2E1 was the primary enzyme in styrene

metabolism, based on inhibition of metabolism in human liver microsome preparations

by the CYP2E1 inhibitor 4-methylpyrazole (Wenker et al. 2001b). These authors also

demonstrated that the maximum velocity (Vmax1) and Michaelis-Menten (Km1) enzyme

kinetics constants varied 6- to 8-fold among 20 human microsomal liver samples;

however, no relationship was found between the interindividual variations in enzyme

kinetics and CYP2E1 polymorphisms.

Assessments of metabolic capacity and interindividual variation in styrene toxicokinetics

in vivo (Wenker et al. 2001c) and stereochemical metabolism of styrene in vivo (Wenker

et al. 2001a) in 20 male volunteers performing physical exercise did not show a

correlation between apparent blood clearance of styrene and individual metabolic

capacity (assessed by administering marker substrates for CYP2E1, CYP1A2, CYP2D6,


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and total CYP450), and only moderate interindividual differences in the stereochemical

metabolism of styrene were observed. Wenker et al. (2001c) concluded that the absence

of a correlation between clearance and metabolic capacity could be due to dependence of

styrene metabolism on liver blood flow.

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Carlson (2004a) reported that wild-type mice were more susceptible to styrene-induced

hepatotoxicity than Cyp2e1-knockout mice, but there was no significant difference in the

response when styrene-7,8-oxide was administered. These results suggest that Cyp2e1 is

important for bioactivation of styrene in the liver. However, Carlson (2003) reported that

there was little or no difference in the rate of metabolism of styrene to styrene-7,8-oxide

by hepatic microsomes from wild-type and knockout mice. Sumner et al. (2001)

compared styrene metabolism in Cyp2e1-knockout and wild-type mice in vivo and

reported that the knockout mice excreted more total urinary metabolites than the wild-

type mice. Carlson (2003) rejected the conclusion that these data indicated that Cyp2e1

was not important in styrene metabolism because this would contradict findings from

many previous studies. Carlson concluded that the data more likely indicate that other

enzymes must be contributing to styrene metabolism in knockout mice. Carlson (2004a)

also noted the reason for this disconnect was unclear, but thought it might be related to

kinetic factors associated with styrene metabolism within the liver of the intact animal.

Carlson (2003), (see Table 2 in Carlson 2003) reported that styrene metabolism by

pulmonary microsomes in Cyp2e1-knockout mice is about one-half that in wild-type

mice. Cyp2f2 was also important for metabolism in mouse lung based on inhibition of

styrene metabolism by the Cyp2f2 inhibitor 5-phenyl-1-pentyne. The same inhibitor

inhibited the pulmonary cytochrome P450 metabolism of styrene in mice in vivo and

prevented an increase in cell replication rates (Green et al. 2001a). Cyp1a and Cyp2b

were considered to play only minor roles in styrene metabolism because of the small

inhibitory effect with α-naphthoflavone, an inhibitor of Cyp1a, and α-

methylbenzylaminobenzotriazole, an inhibitor of Cyp1b (Carlson et al. 1998). Nakajima

et al. (1994b) reported that metabolism by rat lung microsomes was inhibited only by

anti-CYP2B1/2 (which probably corresponds to CYP2B1). In Cyp2e1-knockout studies,

pulmonary toxicity of styrene was similar in both wild-type and knockout mice, which


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supports previous studies that indicated the importance of styrene metabolism by other

enzymes such as Cyp2f2 in the lung (Carlson 2004a).

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Carlson et al. (2001) did not detect 4-vinylphenol when styrene was incubated with

hepatic and pulmonary preparations from rats or mice. However, these tissue preparations

were shown to have considerable 4-vinylphenol metabolizing ability when incubated with

4-vinylphenol in the presence of NADPH. The rate of metabolic activity in mouse liver

microsomes was 3 times faster than in rat liver microsomes, and the rate in mouse lung

microsomes was 8 times faster than in rat lung microsomes. Treatment with pyridine, an

inducer of Cyp2e1, caused a significant increase in 4-vinylphenol metabolism in the liver

but not the lung. Furthermore, 4-vinylphenol metabolism was significantly decreased in

mouse liver and lung microsomes treated with diethyldithiocarbamate, an inhibitor of

Cyp2e1, or 5-phenyl-1-pentyne, an inhibitor of Cyp2f2. This study also indicated that

glutathione conjugation was involved in 4-vinylphenol metabolism, with the highest

activity in mouse lung, with or without the addition of NADPH. Carlson (2002) also

showed that when rats and mice were pretreated with diethyldithiocarbamate or 5-phenyl-

1-pentyne, the hepatotoxicity and pneumotoxicity of 4-vinylphenol were prevented or

greatly decreased. These data suggest that the toxicity of 4-vinylphenol in the liver and

lungs was due to a metabolite rather than to the parent compound. Vogie et al. (2004)

examined the microsomal metabolism of 4-vinylphenol in wildtype and Cyp2e1-

knockout mice and reported no marked differences in the rates of microsomal metabolism

prepared from the lung and liver of mice with either genotype. The knockout mice were

more susceptible to hepatotoxicity than wild-type mice but there was no significant

difference in pneumotoxicity. Thus, in contrast to the findings of Carlson, the animals

that were unable to metabolize 4-vinylphenol through the Cyp2e1 pathway were more

susceptible. Vogie et al. stated that the reason for the discrepancy was unknown, but

could be related to inhibition of other cytochrome P450 enzymes by

diethyldithiocarbamate, or it could have protected against hepatotoxicity by a mechanism

not related to Cyp2e1. The rate of metabolism of 4-vinylphenol metabolism was the same

in wild-type and Cyp2e1-knockout mice indicating that cytochromes P450 other than

Cyp2e1 play an important role (Carlson 2004b). This study also showed that the greatest


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inhibition of enzymatic activity occurred with diethyldithiocarbamate, even in knockout

mice, and suggests that it must inhibit other P450 cytochromes in addition to Cyp2e1.

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5.1.3.6 Detoxification of styrene metabolites 3 Only a very small fraction of the styrene-7,8-oxide formed from styrene in the liver

reaches the systemic circulation. The vast majority is immediately hydrolyzed

enzymatically, as shown by epidemiologic studies (Korn et al. 1994) and based on

theoretical considerations (Arand et al. 1999, Oesch et al. 2000). The hydrolysis of

styrene-7,8-oxide is efficiently carried out by microsomal epoxide hydrolase (mEH).

Although the enzyme has a relatively low turnover, it is able to inactivate genotoxic

substrates very rapidly via formation of a covalent intermediate, an enzyme-substrate

ester, which is subsequently hydrolyzed in a slow, rate-limiting step (Arand et al. 1999,

Tzeng et al. 1998). Overall clearance of styrene-7,8-oxide is efficiently accomplished by

liver mEH; however, detoxification outside the liver is usually less efficient, because it

depends on local mEH levels. Oesch et al. (2000) predicted that the local styrene-7,8-

oxide steady-state level will rise sharply as soon as the rate of styrene-7,8-oxide

formation exceeds the local capacity for enzymatic hydrolysis of styrene-7,8-oxide. As a

result, tissues that produce the relevant CYP and activate styrene to styrene-7,8-oxide in

sufficient amounts, but have low mEH activity, may be more susceptible to styrene-

mediated genotoxicity than would be predicted from the systemic styrene-7,8-oxide load

deduced from the biomarkers measured in blood. The lung, which is the primary entry

site for styrene into the human body, contains cell types that produce styrene-activating

CYP isoenzymes (e.g., Clara cells), but it has significantly lower styrene-activating

activity and mEH activity than the liver.

The second pathway for styrene-7,8-oxide detoxification, the formation of glutathione

conjugates and PHEMAs (which constitute less than 1% of styrene metabolites in

humans) was reported first in rodents (Delbressine et al. 1981) and later in humans

(Maestri et al. 1997). Although GSH conjugation is a minor detoxification route (Maestri

et al. 1997), it may become important in extrahepatic tissues with low mEH activity, such

as lungs and blood-forming organs, as high levels of GST are found in erythrocytes

(Henderson and Speit 2005). Several studies have suggested that polymorphisms in


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GSTM1 may influence metabolite excretion; higher levels of mandelic acid and

phenylglyoxylic acid urinary metabolites (Teixeira et al. 2004) or lower levels of

phenylethyl mercapturic acids (De Palma et al. 2001, Haufroid et al. 2002b) were found

in styrene-exposed GSTM1-null individuals than in wild-type individuals. Studies on

GSTT1 polymorphisms are conflicting (Norppa 2003).

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5.1.4 Excretion 6 The primary route of styrene excretion in both humans and laboratory rodents is the urine

(IARC 1994a); however, the metabolic profiles differ among species (as discussed

above). Almost all of the absorbed styrene is excreted as urinary metabolites; however, a

small fraction (< 5%) may be eliminated as unchanged styrene in exhaled air or urine

(ATSDR 1992, IARC 2002). Mandelic acid and phenylglyoxylic acid (see Table 1-3) are

the primary urinary metabolites, accounting for as much as 95% to 98% of the total

(Manini et al. 2002b). Glutathione conjugates generally account for 1% or less of the

absorbed dose (IARC 1994a).

5.1.4.1 Humans 15 Elimination of styrene from blood was biphasic in human volunteers, indicating a two-

compartment pharmacokinetic process; the half-lives were 0.58 hours for the rapid phase

and 13.0 hours for the slow phase (Ramsey et al. 1980). Urinary elimination of mandelic

acid and phenylglyoxylic acid also were reported to be biphasic in styrene-exposed

workers (IARC 1994a); the half-lives for both were 2.5 hours for the rapid phase and

30 hours for the slow phase (Wieczorek and Piotrowski 1988). Guillemin and Berode

(1988) reviewed data on clearance of these metabolites and also reported that clearances

were biphasic. Half-lives for mandelic acid ranged from 3.9 to 9.4 hours during the first

20 hours post-exposure and from 16.6 to 26.5 hours after 20 hours post-exposure. Half-

lives for phenylglyoxylic acid averaged 10.5 ± 1.4 hours during the first 50 hours post-

exposure in one study reviewed by Guilleman and Berode and ranged from 21.5 to 26.7

during the period from 20 to 200 hours post-exposure in a second study.

IARC (1994a) reported that 0.7% to 4.4% of inhaled styrene was eliminated unchanged

in exhaled air. Unchanged styrene also was reported to be excreted in urine by styrene-


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exposed workers, but the concentration in urine was only about one-tenth that in blood

(Guillemin and Berode 1988, IARC 1994a).

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Pfaffli et al. (1981) reported that they could detect 4-vinylphenol in the urine of styrene-

exposed workers but not in nonexposed individuals by GC/MS, but the level detected was

only 0.3% of the level of mandelic acid in the same individuals. Johanson et al. (2000)

exposed four healthy male volunteers to 50-ppm styrene for 2 hours. Based on the

relationship of mandelic acid and 4-vinylphenol reported by Pfaffli et al. (1981),

Johanson et al. estimated that the maximum level of 4-vinylphenol in their subjects

would be about 0.004 mM [below the detection limit]. Manini et al. (2003) used liquid

chromatography electrospray tandem mass spectrometry to measure 4-vinylphenol

conjugates in urine of workers exposed to styrene and in volunteers exposed to 50 mg/m3

styrene. Urinary 4-vinylphenol conjugates (glucuronates and sulfates) represented about

0.5% to 1% of the total excretion of styrene metabolites and were significantly correlated

with airborne styrene (r = 0.607, P < 0.001) and the sum of mandelic acid and

phenylglyoxylic acid (r = 0.903; P < 0.001) in end-of-shift samples for workers.

5.1.4.2 Experimental animals 16 In rats, elimination of styrene from blood was reported to be biphasic over a period of 6

hours (ATSDR 1992, IARC 1994a). Sumner et al. (1997) reported that after inhalation

exposure to styrene at 250 ppm for 1 to 5 days, male F344 rats, male CD-1 mice, and

male B6C3F1 mice eliminated most of the absorbed styrene in the urine. Following a

single 6-hour exposure, elimination was faster in rats (89% within 12 hours) and CD-1

mice (83% within 12 hours) than in B6C3F1 mice (59% within 12 hours). The slower

elimination in B6C3F1 mice was considered to be consistent with the higher liver toxicity

in these mice. However, when the animals were exposed for 3 to 5 days, elimination in

all three groups was about 88% within 12 hours. The increased excretion in B6C3F1 mice

with longer-term exposure was consistent with induction of styrene metabolism and with

the absence of ongoing acute necrosis following multiple exposures. When CD-1 mice

and male Sprague-Dawley rats were exposed to 14C-labeled styrene by nose-only

inhalation, the primary route of excretion was in the urine (75 ± 7% of inhaled styrene

retained by rats and 63 ± 9% of that retained by mice), and only a small fraction was


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eliminated in the feces of either species (Boogaard et al. 2000a). The species differed in

exhalation of 14CO2, which in two separate experiments accounted for approximately 2%

of retained styrene in rats and 6.4% and 8% in mice. Mice also had higher nonspecific

binding of radiolabeled styrene in nasal passages and lung than rats.

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5.2 Toxicity 5

The toxicity of styrene has been reviewed (ATSDR 1992, Bond 1989, IARC 1994a,

2002). The acute toxicity of styrene in laboratory animals and in humans is considered

low to moderate. The oral LD50 for styrene in rats is 5,000 mg/kg and the inhalation LC50

is 2,770 ppm (2-hour exposure). The LD50 in mice is 320 mg/kg for oral exposure, 660

mg/kg for i.p. injection, and 90 mg/kg for i.v. injection. The inhalation LC50 in mice is

4,940 ppm (4-hour exposure). The major acute toxic effects of styrene include irritation

of the skin and respiratory tract and effects on the central nervous system (CNS).

5.2.1 Humans 13 Drowsiness, listlessness, muscular weakness, and unsteadiness are common signs of

systemic styrene intoxication in humans (Bond 1989). Skin, eye, throat, and respiratory

tract irritation have been reported in styrene-exposed workers (IARC 2002). Direct skin

contact with liquid styrene has caused erythema, dermatitis, and blistering. Minamoto et

al. (2002) conducted patch tests on 29 fiberglass-reinforced-plastics workers. Of the 22

workers who reported skin problems, one had a positive patch test to styrene. In a study

where human volunteers were exposed to styrene concentrations of 51 to 376 ppm for 1

to 7 hours, signs of styrene toxicity (including eye and nasal irritation, nausea, and

headaches) occurred only in subjects exposed to 376 ppm (Bond 1989). In another study

reviewed by Bond, subjects exposed to 800 ppm experienced immediate irritation of the

nose and throat and increased nasal secretions. Respiratory tract irritation was reported in

humans exposed for short durations and airflow restriction in those exposed for long

durations; however, the concentrations and durations were not fully defined. Röder-

Stolinski et al. (2008) investigated the mechanisms responsible for styrene-induced

inflammatory effects using a human alveolar epithelial cell line (A549). Styrene

stimulated the expression of inflammatory mediators, including the chemotactic cytokine

monocyte chemoattractant protein-1 (MCP-1) in these cells. MCP-1 expression and


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glutathione S-transferase [a marker of oxidative stress] was associated with a

concentration dependent pattern of NF-κB activation. NF-κB is a pivotal intracellular

signaling pathway involved in inflammatory responses. Treatment with an NF-κB

inhibitor and an antioxidant were effective in suppressing styrene-induced MCP-1

secretion.

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Respiratory effects from occupational exposure to styrene include bronchitis, asthma, and

pneumonia. Chronic bronchitis has been reported in workers exposed to styrene

concentrations greater than 100 mg/m3 [23 ppm], and increased mortality from

pneumonia was associated with styrene exposure among 40,000 men and women

employed in 660 European reinforced-plastics manufacturing factories; however, no

increased mortality from bronchitis, emphysema, or asthma occurred (IARC 2002). In a

more recent study (reviewed by IARC 2002) of workers in the reinforced-plastics and

composites industry in the United States, there was no relationship between exposure to

styrene and mortality from non-malignant respiratory disease.

Effects of styrene exposure on the nervous system, either central or peripheral, have been

reported mostly for concentrations of 100 ppm or above (IARC 2002). The effects

included decreased nerve conduction velocities and electroencephalographic,

dopaminergic, functional, and psychiatric anomalies. At concentrations below 100 ppm,

reports of effects have been mixed, with some researchers finding no effects and others

reporting memory and neurobehavioral disturbances at concentrations in the range of 10

to 30 ppm. In addition to the effects of styrene on reaction time, color vision, and hearing,

researchers also have studied the possible effects of styrene exposure on taste. However,

Dalton et al. (2003) did not find any evidence for an impairment of olfactory function in

a group of fiberglass-reinforced-plastics workers.

Benignus et al. (2005) conducted a meta-analysis of the relationship of long-term

exposure to styrene and two measures of CNS function: reaction time and color vision.

There was a statistically significant relationship between cumulative exposure to styrene

and an increased choice reaction time as well as an increased color confusion index.

These authors estimated that 8 work-years of exposure to 20-ppm styrene (the ACGIH


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limit) produces a 6.5% increase in choice reaction time and an increase in the color

confusion index equivalent to 1.7 additional years of age in men (the color confusion

index in men increases with age at the rate of about 10% of baseline every 13 years).

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Color vision was reported to be impaired in several studies reviewed by IARC (2002),

and it was proposed that this effect reflects changes in neural functioning along optic

pathways. Effects were seen at concentrations of styrene as low as 20 ppm; however, one

study reported that the effects of styrene on color vision were reversed after 4 weeks in a

styrene-free environment. In a study of 108 workers in Swedish reinforced-plastics

plants, Iregren et al. (2005a) concluded that there was a “strong indication” that color

vision was negatively affected in workers with exposure below the Swedish occupational

exposure limit of 90 mg/m3 [21 ppm]. Confounding effects of age and higher past

exposure levels were also considered by the authors, but they did not consider these

factors sufficient to explain all of the differences observed.

Significant changes in hearing thresholds at high frequencies have been reported in

workers exposed to styrene in some studies (IARC 2002). Although several studies

reviewed by IARC did not find effects on hearing threshold at styrene concentrations

below 150 mg/m3 [35 ppm], one study reported disturbances in the central auditory

pathways in 7 of 18 workers exposed for 6 to 15 years to styrene at concentrations below

110 mg/m3 [25 ppm]. Several recent publications have reviewed the relationship between

styrene exposure and hearing loss. Hoet and Lison (2008) reported that styrene appears to

be ototoxic in rats, but the human data were insufficient to support a clear conclusion.

Sliwinska-Kowalska et al. (2007) reported the findings of a scientific workshop that

reviewed the ototoxic effects of organic solvents. Seven of nine occupational studies of

styrene-only exposure (primarily in the glass fiber–reinforced-plastics industry) showed

evidence of hearing loss. Measurements varied among the studies but included pure tone

audiometry, high-frequency hearing loss, and central hearing tests. Although one of the

primary conclusions from the workshop was that styrene is a risk factor for hearing loss,

the authors concluded that the data were not sufficient to derive a dose-response

relationship. Johnson (2007) also reviewed these nine studies and noted that the reported

effects occurred at concentrations below the current TLV values (20 to 50 ppm) but the


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authors considered the effects to be negligible. Fuente and McPherson (2006) also

reviewed the literature on solvent exposure and hearing loss. They reported that a positive

linear relationship was seen between an average working-life exposure to styrene and

hearing thresholds at 6,000 and 8,000 Hz. These authors also noted that there was an

additive effect on hearing thresholds with exposure to both styrene and noise. Johnson et

al. (2006) reported audiological findings in 313 workers from fiberglass and metal-

product manufacturing plants. Workers exposed to noise and styrene had significantly

poorer pure-tone thresholds in the high-frequency range than controls or noise-only

exposed workers.

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Toppila et al. (2006) noted that styrene is both ototoxic and neurotoxic; thus, styrene

exposure could affect postural stability. These authors investigated the effects of low

concentrations of styrene on postural stability among 252 male Finnish fiberglass-

reinforced plastic boat manufacturers. Smoking history, postural stability, and urine

mandelic and phenylglyoxylic acid concentrations were determined. Breathing zone

measurements of styrene were measured for 148 workers. Mean styrene concentrations

for the age-matched workers were 21 mg/m3 [4.8 ppm] for nonlaminators and 108 mg/m3

[25 ppm] for laminators. Their analysis included 88 matched pairs and indicated that

postural stability among boat laminators was impaired compared with nonlaminators. The

impairment occurred among young workers and worsened with age.

Hepatic and renal effects of styrene exposure were mixed or absent in older studies, but

more recent reports have found alterations in hepatic clearance of bilirubin and in hepatic

alanine and aspartate transaminase activities (IARC 2002). Urinary markers for renal

toxicity are reported to be only weakly correlated with styrene exposure.

The early studies that examined the effects of styrene exposure on the hematopoietic and

immune systems failed to find consistent functional changes (IARC 2002). One study

found no differences in hemoglobin, erythrocyte, and leukocyte concentrations in 84

workers exposed to styrene concentrations of 50 to 500 ppm for 1 to 36 years. Another

study found no evidence of hematological abnormalities in 163 workers in a styrene-

butadiene synthetic rubber manufacturing plant. More recent studies reviewed by IARC


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reported a 30% increase in the number of peripheral blood monocytes in workers exposed

to 13-ppm styrene, and an exposure-related decrease in both the mean corpuscular

hemoglobin and neutrophil concentrations among 221 workers in the reinforced-plastics

industry that were exposed to 1 to 100 ppm for 1 to 20 years. Other studies reported

effects on the immune system, including a reduction in total T lymphocytes and T-helper

lymphocytes along with an increase in natural killer cells, and alterations in the cell-

mediated immune response of T lymphocytes. Changes in lymphocyte subpopulations

were observed mainly at concentrations greater than 50 ppm. Biro et al. (2002)

investigated the immunotoxicity of styrene in 10 styrene-exposed workers compared with

29 healthy controls. The data indicated that changes in the expression of surface antigens

on peripheral lymphocytes were correlated with exposure. The styrene-exposed group

had a significant decrease in the level of CD25+ CD4+ lymphocytes (activated helper T

cells) with a concomitant increase in the level of CD45RO+ CD4+ lymphocytes (memory

helper T cells), suggesting a shift from activated to memory helper T cells. The styrene-

exposed group also had a slightly higher ratio of CD4+ lymphocytes to CD8+ T

lymphocytes, which the authors concluded was caused by smoking as this was seen

among all smokers compared with nonsmokers, and the styrene-exposed group had a

higher percentage of smokers than the control group,

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Plotnick and Weigel (1979) suggested that a relationship may exist between the

distribution of styrene and/or its metabolites in the pancreas and the increased glucose

tolerance reported in workers. Chmielewski (1976) reported a statistically significant

increased glucose tolerance in workers exposed to styrene for 1 year; however, the

increase was not significant in workers exposed for 10 years. Some epidemiological

studies have reported pancreatic cancer in workers exposed to styrene (see Section 3.8.2).

Impaired glucose metabolism (diabetes mellitus) has been observed in patients with

pancreatic cancer, although it is not known whether diabetes develops shortly before or

after the clinical manifestations of pancreatic cancer. A meta-analysis of 11 case-control

studies (including only studies in which diabetes was present at least 1 year before

diagnosis of pancreatic cancer) and 9 cohort studies found a relative risk of 2.1 (95% CI

= 1.6 to 2.8) for pancreatic cancer in diabetics, compared with non-diabetics (Everhart

and Wright 1995). A large prospective cohort study found an association between post-


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load plasma glucose concentration and pancreatic cancer in individuals without self-

reported diabetes, which the authors considered suggestive that factors associated with

abnormal glucose metabolism could play a role in development of pancreatic cancer

(Gapstur et al. 2000). In a review of pancreatic cancer, Michaud (2004) concluded that

chronic pancreatitis and diabetes mellitus are medical conditions that have been

consistently related to pancreatic cancer, and the evidence suggesta that they are

causually related to pancreatic cancer rather than consequences of the cancer.

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Matanoski and Tao (2003) reported an association between cardiovascular disease and

occupational exposure to styrene. Their case-cohort study included 498 cases that died

from ischemic heart disease and a 15% random sample (N = 997) of all male workers

who were employed during 1943 to 1982 in two styrene-butadiene rubber manufacturing

plants in the United States. Recent styrene exposure was significantly associated with

acute ischemic heart disease death among active workers. The relative hazard of death for

exposure during the most recent two years among active workers with 2 or more years of

employment was 2.95 (95% CI = 1.02 to 8.57) at a time-weighted styrene concentration

of 0.2 to < 0.3 ppm and 4.3 (95% CI = 1.56 to 11.84) at time-weighted exposure

concentrations of ≥ 0.3 ppm. Delzell et al. (2005) also examined the relationship of

styrene exposure and mortality from ischemic heart disease among 16,579 men employed

at 6 styrene-butadiene rubber manufacturing plants (including the 2 plants reported by

Matanoski and Tao) for at least one year and employed from 1943 until 1990. Men in the

highest quintile of exposure (> 5.5 ppm) and in the highest quintile of cumulative

exposure (> 60.67 ppm-year) had ischemic heart disease ratios of 1.14 (95% CI = 0.96 to

1.35), and 1.06 (95% CI = 0.90 to 1.27), respectively. Acute disease was not associated

with average intensity of exposure within the most recent 2 years. Incidences of chronic

disease were elevated in subjects with the highest exposure, but the associations were

weak and imprecise, and there was limited evidence of a dose-response relationship. The

authors concluded that their study did not provide strong support for a causal association

between styrene and mortality from ischemic heart disease.

The potential reproductive and developmental effects of styrene in humans have been

reviewed (IARC 2002, NTP 2006). Some earlier studies suggested an association


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between occupational exposure to styrene and congenital CNS malformation and

spontaneous abortions; however, these associations were not confirmed in later studies

(IARC 2002). Other studies have not shown a consistent or statistically significant

relationship between styrene exposure and reduced birth weight, sperm abnormalities,

time-to-pregnancy, or menstrual cycle effects. NTP (2006) concluded that the human data

were insufficient to conclude that styrene is a reproductive or developmental toxicant;

however, based on the animal data, the panel expressed negligible concern for effects in

humans. There was suggestive evidence that occupational exposure to styrene was

associated with increased serum prolactin and depletion of peripheral blood dopamine-

metabolizing enzyme activities, but the clinical relevance of these findings was unclear.

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Several publications have reported increased serum prolactin levels in workers exposed

occupationally to styrene (Arfini et al. 1987, Bergamaschi et al. 1996, Bergamaschi et al.

1997, Mutti et al. 1984); a proposed cause is dopaminergic dysfunction resulting from the

interaction between styrene metabolites and dopamine (Mutti and Smargiassi 1998).

Although the relationship between the possible styrene-related increases in serum

prolactin and breast cancer is not known, Harvey (2005) concluded that the evidence for

the role of prolactin in human breast cancer is strong and consistent. Several large

epidemiology studies have shown that dopamine antagonists increase breast cancer risk.

Hyperprolactinemia is associated with human breast cancer growth and poor prognosis,

and prolactin is a mitogen in human breast cancer cells that suppresses apoptosis and

upregulates BRCA1. An increased risk of breast cancer was not observed in the cohort

studies of styrene-exposed workers, which consisted predominantly of men; however,

two studies of the general population (a case-control study and an ecological study)

reported an association between breast cancer and styrene exposure (see Sections 3.5.2

and 3.8.6).

5.2.2 Experimental animals 26 The toxic effects of styrene in experimental animals have been reviewed (ATSDR 1992,

Bond 1989, IARC 2002). Acute exposures were associated with eye and nose irritation,

CNS depression, and death at high concentrations. Subacute to subchronic exposures

have been associated with adverse effects on the liver, pancreas, kidney, nervous system,


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respiratory system, immune system, and hematopoietic system. This section briefly

reviews the overall toxicity in experimental animals (5.2.2.1) and then describes in

greater detail studies of respiratory toxicity (5.2.2.2), toxicity of the stereoisomers of

styrene-7,8-oxide (5.2.2.3), and glutathione depletion (5.2.2.4), because these factors

have been suggested to play a role in the development of lung tumors in mice (see

Sections 5.3 and 5.5).

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5.2.2.1 Overall toxicity findings 7 Acute exposures of rats and guinea-pigs to styrene at a concentration of 650 ppm resulted

in eye and nose irritation (IARC 2002). Higher concentrations resulted in weakness,

unsteadiness, and other CNS effects (1,300 ppm), unconsciousness (2,500 ppm) and

death (5,000 to 10,000 ppm). Eye and nose irritation was also reported in rats exposed to

styrene concentrations of 1,300 or 2,000 ppm for 7 to 8 hours per day, 5 days per week

for about 6 months (Spencer et al. 1942, Wolf et al. 1956).

Permanent hearing loss, neurotoxic effects, hematopoietic and immune system effects,

and damage to the pancreas, lung, liver, and kidney have been reported in rats, mice, or

guinea-pigs (IARC 2002). Sliwinska-Kowalska et al. (2007) reported that the lowest

concentration of styrene known to cause hearing loss in rats is 300 ppm. Styrene damages

the outer hair cells in the cochlea. The neurotoxic effects included decreased monoamine

oxidase activity, depletion of dopamine, weakness, and brain damage. Gagnaire et al.

(2006) investigated the effects of styrene on the extracellular and tissue levels of

dopamine, serotonin, and their metabolites in male rats exposed to 750- or 1,000-ppm

styrene for 4 weeks. Rats exposed to the high dose had a significant decrease in

extracellular acid metabolite concentrations, while tissue levels of these metabolites were

decreased to a lesser extent. The effects were reversed after 17 days. Umemura et al.

(2005) investigated the neuroendocrinological effects in rats exposed to 150-ppm styrene

for 10 days. The styrene concentration in the blood was higher in female rats than in male

rats, and the prolactin level was significantly increased in female rats. Levels of

neurotransmitters were not affected in either sex; therefore, the mechanism enhancing

prolactin secretion was unclear. Mouse splenic T-lymphocyte activity was suppressed by

in vitro exposure to styrene, and oral dosing (20 to 50 mg/kg b.w. styrene daily for five


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days) impaired humoral and cell-mediated immunity in male Swiss mice (IARC 2002).

The number of erythropoietic cells was increased in male Sprague-Dawley rats exposed

to styrene by inhalation or i.p. injection. Nano et al. (2000) exposed groups of 6 male

Sprague-Dawley rats to i.p. injections of styrene at 40 or 400 mg/kg b.w. or corn oil for 3

consecutive days or by inhalation of styrene vapor (purity 99%) at 0 or 300 ppm 6

hours/day, 5 days/week for 2 weeks. Some of the rats (inhalation exposure) were killed

immediately after the last treatment while the others were killed 3 weeks later. Rats

injected with 400 mg/kg styrene showed hyperactivity of the erythropoietic series while

the granulocytopoietic series was normal. There was a statistically significant increase in

basophilic, polychromatophilic, and orthochromatic erythroblasts in rats that inhaled

styrene for 2 weeks. There also was a temporary block of immature cells of the

granulocytopoietic series.

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Khanna et al. (1994) exposed mice, rats, and guinea-pigs to styrene orally in groundnut

oil on 5 days per week for 4 weeks at 25 or 50 mg/kg b.w. per day for mice and 160 or

320 mg/kg b.w. per day for rats and guinea-pigs. Mice exhibited moderate inflammatory

reaction of pancreatic islet cells, congestion of pancreatic blood vessels, moderate

congestion of pancreatic lobules, and increased serum insulin levels. Guinea-pigs showed

congestion of pancreatic acinar parenchyma, marked degranulation of beta cells of large

pancreatic islets, and decreased serum insulin levels. No changes in the pancreas were

noted in rats other than decreased serum insulin levels, and no significant changes in

blood glucose levels were noted in any of the species studied.

Subacute to subchronic exposure to styrene by i.p. injection or inhalation has caused

kidney and liver damage in rodents (IARC 2002). These effects were often associated

with glutathione depletion. B6C3F1 mice exposed for 14 days to styrene by inhalation at

a concentration of 0, 125, 250, or 500 ppm developed severe centrilobular hepatic

necrosis (Morgan et al. 1993c, Morgan et al. 1993b). Mortality was higher in the 250-

ppm group of each sex than in the 500-ppm group. The differences in mortality could not

be explained on the basis of styrene-7,8-oxide production, GSH depletion, or

hepatotoxicity. Sprague-Dawley rats given repeated i.p. injections of 2.9 to 5.8 mg/kg

b.w. for 6 weeks had morphological changes in the kidney (IARC 2002). Mild tubular


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damage occurred in Sprague-Dawley rats given daily i.p. injections of 1 g/kg b.w. for 10

days, and inhalation exposure to 300 ppm for 12 weeks resulted in a slight increase in the

urinary excretion of plasma proteins and minor changes in kidney histopathology.

Hepatotoxic effects included focal necrosis in male albino rats exposed orally to 400

mg/kg b.w. styrene for 100 days. Centrilobular necrosis was reported in several studies in

mice exposed to 125 to 500 ppm for 2 weeks. Sex and strain differences in sensitivity

have not generally correlated with differences in glutathione depletion or concentrations

of styrene or styrene-7,8-oxide in blood. Single i.p. injections of 250 to 1,000 mg/kg b.w.

of styrene or styrene-7,8-oxide produced a dose-dependent increase in serum sorbitol

dehydrogenase activity [an indicator of hepatotoxicity]. One study indicated that liver

toxicity was greater when styrene was administered by i.p. injection compared with

inhalation of styrene vapor (De Piceis Polver et al. 2003). This may be explained by the

fact that the intraperitoneal route results in direct exposure of the liver. Several studies

have shown that hepatotoxicity may be enhanced in mice pretreated with CYP enzyme

inducers. In one study, the hepatotoxic effects of styrene-7,8-oxide were increased in

mice by administration of trichloropropene oxide, an inhibitor of epoxide hydrolase.

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The developmental and reproductive toxicity of styrene in experimental animals have

been reviewed (Brown et al. 2000, IARC 2002, NTP 2006). The available studies have

not shown an increased incidence of malformations, but there have been reports of

increased embryonic, fetal, and neonatal deaths; skeletal and kidney abnormalities;

decreased birth weight, postnatal developmental delays (e.g., incisor eruption, eye

opening), and neurobehavioral and neurochemical abnormalities. The reported effects

were seen mostly at high doses that were associated with maternal toxicity, but at least

one study indicated that styrene might affect the developing brain and postnatal

development. Beliles et al. (1985) conducted a three-generation study of the reproductive

effects of styrene exposure in Sprague-Dawley rats (see Section 4.2.1). The animals were

exposed to styrene indirectly in utero and as neonates, and directly in drinking water

while maturing to become breeders. Fertility, litter size, pup viability, pup survival, sex

ratio, pup body weight, weanling kidney and liver weight, and marrow cytogenetics were

evaluated. The only reported effects included an apparent reduction in 21-day survival of

high-dose F1 pups, and a loss of breeding efficiency in F3 parents; however, the authors


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noted that there were mitigating factors. These effects were not consistent and were

traced to a single or only two individual animals or litters. The authors concluded that

styrene exposure produced no deleterious dose-related effects or decrements in

reproductive function through three generations.

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5.2.2.2 Respiratory toxicity 5 Respiratory toxicity, including nasal toxicity and pneumotoxicity, has been observed in

mice exposed to styrene or styrene-7,8-oxide. The Clara cell is the main site of both

bioactivation and toxicity of styrene in the lung (Harvilchuck and Carlson 2006, Hynes et

al. 1999).

Green et al. (2001b, 2001a) exposed CD-1 mice to styrene at 40 or 160 ppm for 3 days.

Styrene exposure caused degenerative changes in the nasal cells (including atrophy of the

olfactory mucosa and loss of normal cellular organization) and pneumotoxicity

(characterized by focal loss of cytoplasm and focal crowding of nonciliated Clara cells,

particularly in the terminal bronchiolar region). In mice exposed for 3 days or longer, cell

replication rates were increased in the terminal and large bronchioles (Green et al.

2001a). Similar effects occurred in mice given oral doses of 100 or 200 mg/kg for 5 days,

but not in rats exposed to 500 ppm for up to 10 days. There were no morphological or

cell proliferation effects in the alveolar region of the mouse lung. Female CD-1 mice

exposed to styrene at 40 or 160 ppm for 1 to 20 consecutive days had decreased levels of

Clara-cell–specific protein (CC16) in lung lavage fluid and blood serum, suggesting

destruction of Clara cells (Gamer et al. 2004). Swiss-Albino mice given i.p. injections of

styrene at 800 mg/kg b.w. or styrene-7,8-oxide at 300 mg/kg b.w. had increased levels of

gamma-glutamyltranspeptidase (GGT) and lactate dehydrogenase (LDH) in the

bronchioalveolar lavage fluid [these are markers of pneumotoxicity] (Gadberry et al.

1996).

Cruzan et al. (1997) reported nasal toxicity (atrophy of the olfactory epithelium and

olfactory nerve fibers, with or without focal respiratory metaplasia) and lung toxicity in

CD-1 mice exposed to styrene at a concentration of 100, 150, or 200 ppm for 13 weeks;

at 50 ppm, only nasal toxicity was seen. Changes in the lung included decreased

eosinophilia of the bronchial epithelium, focal crowding of nonciliated cells in


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bronchioles, and focal bronchiolar epithelial proliferation. An increased labeling index in

Clara cells was observed after two weeks, but no increase was observed in type II

pneumocytes; however, the authors reported that the labeling index in the cell

proliferation studies was highly variable among rodents in the same exposure group.

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In the chronic inhalation study (see Section 4.1.2), styrene exposure (20, 40, 80, or 160

ppm) resulted in toxic effects in the nasal passages and lung of CD-1 mice (Cruzan et al.

2001). Histological effects in the nasal passages included respiratory metaplasia of the

olfactory epithelium with changes in the underlying Bowman’s glands and loss of

olfactory nerve fibers. The effects increased in severity with increasing styrene

concentration and duration of exposure, and most changes were observed in all exposure

groups by 78 weeks. In the lung, styrene exposure resulted in decreased eosinophilic

staining of Clara cells at 12, 18, and 24 months. Bronchiolar epithelial hyperplasia was

observed at 12 months (at concentrations > 40 ppm) or 18 months (at 20 ppm); the

hyperplasia extended into the alveolar ducts in the high-dose animals. (Lung tumors were

observed after 24 months, see Section 4.1.2)

Respiratory toxicity has also been reported in rats exposed to styrene. Exposure at 150 or

1,000 ppm caused a dose-related decrease in tracheal and nasal ciliary activity, but at 12

weeks post exposure, ciliary activity had returned to near control values in the low-dose

group and to 50% to 75% of control values in the high-dose group (Ohashi et al. 1986).

Epithelial changes in the nose and trachea (vacuolation of epithelial cells, nuclear

pyknosis, and exfoliation of epithelial cells) were observed in rats exposed to styrene at

800 ppm (IARC 2002). Cruzan et al. (1997) also reported histological changes in the

olfactory epithelium (focal disorganization, focal hyperplasia of basal cells, single-cell

necrosis, and cell loss) in CD (Sprague-Dawley-derived) rats exposed to styrene by

inhalation at 500 to 1,500 ppm for 13 weeks. Coccini et al. (1997) reported cytoplasmic

changes involving bronchiolar and alveolar type II cells (similar to those observed in

mice) in Sprague-Dawley rats exposed to styrene by either i.p. injection (40 or 400 mg/kg

b.w. daily) or inhalation (300 ppm for 2 weeks); damage was more severe following i.p.

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In contrast, several studies have not detected pneumotoxicity in rats. Gamer et al. (2004)

reported no signs of lung toxicity in female CD rats exposed to styrene at up to

2,150 mg/m3 [490 ppm] for up to 21 days, and Green et al. (2001a) did not observe

morphological or cell-proliferative changes in the lungs of Sprague-Dawley rats exposed

at 500 ppm for up to 10 days; however, lung toxicity was observed in mice in these

studies (see above). Cruzan et al. (1997) also did not observe lung toxicity or increased

cell proliferation in CD rats [although there was a high variability in the percentage of

cells labeled with bromodeoxyuridine].

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Some studies have suggested that styrene metabolites other than styrene-7,8-oxide also

cause cytotoxicity in the lung. Cruzan et al. (2002) reported that styrene metabolism in

mice produced 4- to 10-fold more metabolites via ring-oxidation and the

phenylacetaldehyde pathways than observed in rats. In another study, the toxicity of 4-

vinylphenol, a ring-oxidized metabolite of styrene, was evaluated in lungs of CD-1 mice

and female Sprague-Dawley rats exposed by i.p. injection in 3 daily divided doses (2, 6,

20, or 60 mg/kg b.w. per day) for 14 consecutive days (Cruzan et al. 2005a). Multifocal

hyperplasia was present in the medium bronchi and terminal bronchioles in some of the

mice exposed to 6 or 20 mg/kg b.w. and in all of the mice in the high-dose group.

However, no evidence of toxicity was found in the lungs of Sprague-Dawley rats. Several

studies have investigated the metabolism of the styrene metabolite 4-vinylphenol in rat

and mouse liver and lung. Carlson et al. (2002) concluded that 4-vinylphenol is a more

potent hepatotoxicant and pneumotoxicant than either styrene or styrene-7,8-oxide based

on increases in SDH (a marker for hepatic toxicity) in serum and increases in cell

numbers and LDH levels in bronchoalveolar lavage fluid from adult male CD-1 mice

injected i.p. with 50 mg/kg 4-vinylphenol compared with doses of 500 to 1,000 mg/kg for

styrene and 300 mg/kg styrene oxide to induce significant effects in separate experiments

from their laboratory in another strain of mice (non-Swiss Albino) (Gadsberry et al.

1996).

Kaufmann et al. (2005) investigated the effects of styrene and its metabolites on the

mouse lung. CD-1 mice were injected i.p. with styrene, styrene-7,8-oxide, 4-vinylphenol,

1-phenylethanol, 2-phenylethanol, phenylacetaldehyde, phenylacetic acid, or


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acetophenone. Of the compounds tested, only styrene-7,8-oxide (at 100 mg/kg b.w. 3

times per day) and 4-vinylphenol (5, 20, or 35 mg/kg b.w. 3 times per day) caused

increases in cell proliferation in large/medium bronchi (up to 15.1 fold for 4-vinylphenol

and 7.5 fold for styrene-7,8-oxide) and terminal bronchioles (up to 19.7 fold for 4-

vinylphenol and 10.5 fold for styrene-7,8-oxide). Both compounds also caused

glutathione depletion and histomorphological changes in the bronchiolar epithelium.

These two molecules also caused histopathological changes in the terminal bronchioles

that included the appearance of flattened cells and the loss of the typical bulging of the

apical cytoplasm of Clara cells (which the authors describe as “dome-shaped”) into the

bronchial lumina. Styrene-7,8-oxide, but not 4-vinylphenol, also caused marginal

increases in alveolar cell proliferation and an increased number of apoptotic cells in

large/medium bronchi. Kaufmann et al. concluded that the metabolites of the side-chain

hydroxylation pathway (phenylethanols, acetophenone, phenylacetaldehyde, and

phenylacetic acid) were of minor relevance for the pneumotoxic effects in the terminal

bronchioles, and they proposed that ring-oxidized metabolites could be the cause of

styrene-induced oncogenicity based on the cytotoxicity of the ring-oxidized metabolite 4-

vinylphenol for Clara cells and the resulting proliferative response in the terminal

bronchioles.

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Chung et al. (2006) compared the cytotoxicity of styrene and styrene-7,8-oxide in a

transgenic cell line expressing CYP2E1 (h2E1) and the wild-type cell line (cHol, human

B-lymphoblastoid). Cell viability assays demonstrated that styrene was toxic to h2E1

cells (IC50 = 121.8 μM) but no significant increase in cell death was observed in wild-

type cells at concentrations as high as 1,000 μM. However, there was no significant

difference in susceptibility of h2E1 and wild-type cells exposed to styrene-7,8-oxide.

These data indicate that CYP2E1 and styrene-7,8-oxide have an important role in the

cytotoxic effects of styrene in these cell lines. Inhibition of epoxide hydrolases enhanced

cytotoxicity while glutathione conjugation reduced cytotoxicity.

5.2.2.3 Toxicity of styrene stereoisomers 28 Gadberry et al. (1996) examined the pneumotoxicity and hepatotoxicity of styrene and

styrene-7,8-oxide (the racemic mixture and R- and S-enantiomers) in adult male non-


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Swiss albino mice. GGT and LDH activity in bronchioalveolar lavage fluid and the

activity of the hepatic enzyme serum sorbitol dehydrogenase (SDH) were measured.

Groups of 8 to 10 mice were sacrificed 24 hours after i.p. injection with 300 mg/kg b.w.

of either racemic, R-, or S-styrene-7,8-oxide or 800 mg/kg b.w. of styrene, and another

group of mice was sacrificed 6 hours after i.p. injection with 300 mg/kg b.w. of either

racemic, R-, or S-styrene-7,8-oxide. Data for the 24-hour sacrifice are shown in Figure 5-

2. The R-isomer of styrene-7,8-oxide was more hepatotoxic than the S-isomer at both the

6-hour (data not shown) and 24-hour time points, based on a significant (P < 0.05)

increase in SDH activity. In the tests for pneumotoxicity (GGT and LDH), enzyme

activity was higher at both time points in the lungs of mice administered the R-isomer

than in those administered the S-isomer. The results for the racemic mixture were

variable, being sometimes higher than for either individual isomer, sometimes lower, and

sometimes intermediate. However, the authors reported that none of the differences were

statistically significant.

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Figure 5-2. Pneumotoxicity and hepatotoxicity of styrene-7,8-oxide enantiomers in male non-Swiss albino mice at 24 hours after i.p. administration

Source: adapted from Gadberry et al. 1996. Results for exposure groups with different letters (a, b) differed significantly from each other at P < 0.05.


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In an in vitro study using isolated Clara-cell (~90% of total cells) preparations prepared

from lungs of adult male CD-1 mice, Harvilchuck and Carlson (2006) reported that

styrene (LC50 = 1.721 mM) was the most toxic substance tested, followed by racemic

styrene-7,8-oxide (2.344 mM), R-styrene-7,8-oxide (3.243 mM), 4-vinylphenol (3.500

mM), and S-styrene-7,8-oxide (4.842 mM); [however, no statistical comparisons were

reported and the toxicity data for the various compounds appeared to overlap in the

published graph]. Clara cells isolated from Sprague-Dawley rats were 4-fold less

susceptible to cytotoxicity of styrene and its metabolites than mouse Clara cells. Styrene

also was the most toxic compound tested in isolated rat Clara cells with 4-vinylphenol

and S-styrene-7,8-oxide being the least toxic. Incubation of mouse Clara cells with test

agents at 0.1, 0.5, and 1.0 mM concentrations resulted in significantly (P < 0.05, Student-

Newman-Keul’s test) decreased glutathione levels after a 3-hour incubation with the

following substances and concentrations: styrene (1.0 mM), racemic styrene-7,8-oxide

(0.5 or 1.0 mM), R-styrene-7,8-oxide (1.0 mM), S-styrene-7,8-oxide (1.0 mM), and 4-

vinylphenol (0.5 or 1.0 mM). In in vivo experiments, racemic and R-styrene-7,8-oxide

(300 mg/kg b.w. doses for each) significantly (P < 0.05, Student-Newman-Keul’s test)

decreased Clara-cell glutathione concentrations at 3 hours after intraperitoneal injection

of test agents compared with corn-oil controls, while neither styrene (600 mg/kg b.w.), S-

styrene-7,8-oxide (300 mg/kg b.w.), nor 4-vinylphenol (100 mg/kg b.w.) differed

significantly from controls at this time point.

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Several studies have shown differential genotoxicity of the styrene-7,8-oxide

enantiomers, in the following order of mutagenicity: R-enantiomer > racemic mixture >

S-enantiomer (Pagano et al. 1982, Seiler 1990). However, chromosomal aberrations and

sister chromatid exchange in mouse bone marrow cells were increased significantly

following in vivo exposure to the S-isomer but not the R-isomer, and the mitotic index

was decreased significantly for both isomers (Sinsheimer et al. 1993). (See Section

5.5.2.4 for a more detailed discussion of these studies).

5.2.2.4 Gluthathione depletion 28 Depletion of GSH in the lung has been reported to be associated with an increased risk of

lung damage and disease (Rahman et al. 1999), and GSH depletion generally has been


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shown to correlate with chromosomal DNA fragmentation associated with apoptosis and

necrosis (Higuchi 2004). Although Cohen et al. (2002)

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6 reported that both measured

GSH concentrations and those predicted by a physiologically based pharmacokinetic

(PBPK) model indicated a greater decrease in GSH in the lungs of mice than rats, these

effects were seen only at styrene concentrations of 40 ppm or greater, which exceed

concentration of 20 ppm at which hyperplasia in the mouse lung was reported by Cruzan

et al. (2001). Also, Gamer et al. (2004) reported that 20 exposures (6 hours per day, 5

consecutive days per week, for 4 weeks) of female CD-1 mice to styrene at 160 ppm,

which produced cellular crowding in the epithelial lining of the lung, indicative of very

early hyperplasia, did not increase concentrations of 8-hydroxy-deoxyguanosine. No

evidence of oxidative stress was seen despite depletion of GSH in homogenates from the

styrene-exposed mouse lungs. Similarly exposed female Crl:CD rats did not show any

signs of lung toxicity. Turner et al. (2005) also found a decrease in GSH in the lungs of

mice exposed by i.p. injection to styrene (600 mg/kg b.w., or 5.8 mmol/kg) and styrene-

7,8-oxide (300 mg/kg b.w., or 2.5 mmol/kg). However, administration of 4-vinylphenol

(100 mg/kg, or 0.83 mmol/kg), which is a more potent hepatotoxin and pneumotoxin than

styrene or styrene-7,8-oxide, caused less depletion of GSH.

5.2.3 Estrogenicity studies 18 Several studies have been published on the potential estrogenicity of polystyrene

oligomers, which can leach from polystyrene food containers. Polystyrene dimer and

trimer extracts from food containers were tested in vitro for estrogen-like effects using

estrogen-responsive element reporter, estrogen receptor binding, and cell-proliferation

assays, and in vivo using a rat uterotrophic assay. Bachman et al. (1998) measured the

effect of extracts from 23 polystyrenes in a rat uterotrophic assay at concentrations up to

0.75 mg/L [equivalent to 15 microgram/kg-b.w. per day]. None of the polystyrene

extracts were positive in this assay. Fail et al. (1998) measured the estrogenicity of a

polystyrene extract equivalent in dose per body weight to human consumption [amount

not specified]. It was negative in a rat uterotrophic assay and in an estrogen-responsive

element reporter assay [3 mM, approximate maximum concentration of polystyrene



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extract tested]. Azuma et al. (2000) and Date et al. (2002) reported a lack of estrogenicity

of styrene monomers, dimers and trimers using in vivo and in vitro assay systems. Ohno

et al. (2001) used high concentrations [up to 10-3 M in vitro] of specific oligomers in

uterotrophic, estrogen-responsive element reporter, and estrogen receptor binding assays

and also obtained negative results. In this study, styrene monomer, three styrene dimers,

and seven styrene trimers known to dissolve in small amounts from polystyrene cup

noodle containers were tested. However, Ohyama et al. (2001) tested the same styrene

dimers and trimers and obtained positive results (2 positives out of 4 dimers tested and 4

positives out of 7 trimers tested) at concentrations of 10-6 and 10-5 M [highest

concentration tested] in a cell-proliferation assay and in a binding affinity assay for

human estrogen receptor alpha (9 oligomers were positive and 2 trimers were negative in

this assay). These results were refuted by Ohno et al. (2003), whose laboratory tested the

same oligomers from the Ohyama report using three different estrogen receptor binding

assays. The results for all oligomers were negative in these assays. Further, the results of

a rat uterotrophic assay and estrogen response element reporter assay were also negative

using the same styrene oligomers. In a letter to the journal Environmental Health

Perspectives, Ohno and colleagues (Ohno et al. 2002) noted that in the assay system of

Ohyama, solubility was a problem at high concentrations leading to false positive results,

and the validity of the MCF-7 E-Screen assay was also questioned. Ohyama and Nagi

replied that their results were valid, because they believed the insolubility of styrene

oligomers observed in Ohno and colleagues’ studies appeared to be due to using water

rather than DMSO (as was used in the Ohyama studies) to dissolve the compounds.

Ohyama also defended the use of the MCF-7 E-screen method as a well recognized

method for estrogenic screening.

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It is possible that metabolic activation of styrene oligomers may affect the estrogenicity

of these compounds. Kitamura et al. (2003), using rat liver microsomes as a metabolic

activating system, found the activated form of trans-1,2 diphenylcyclobutane, a styrene

dimer, to be estrogenic using a yeast estrogen screening assay and an estrogen-responsive

element reporter assay. The active metabolite was a hydroxylated form called trans-1(4-

hydroxyphenyl)2-phenylcyclobutane [activity at 10-5M]. According to the authors, cis-

1,2-diphenylcyclobutane, 1,3-diphenylpropane, and 2, 4-diphenyl-1-butene also exhibited


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estrogenic activity after metabolic activation, but the activity was lower than with cis-1,2-

diphenylcyclobutane.

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5.3 Interspecies differences in metabolism, toxicity, and toxicokinetics 3 In its summary of styrene exposure studies in humans (volunteers or workers) and

experimental animals, IARC (2002) stated that toxicokinetic pathways are qualitatively

similar in humans and animals, but differ quantitatively. This section reviews studies and

toxicokinetic models of interspecies differences in styrene-7,8-oxide formation,

stereochemistry, and metabolism of styrene-7,8-oxide.

5.3.1 Styrene-7,8-oxide formation in the lung 9 Styrene-7,8-oxide, a primary metabolite of styrene, is considered to cause many of the

toxic and genotoxic effects resulting from styrene exposure, including those in the lung.

Clara cells are primarily responsible for the metabolism of styrene to styrene-7,8-oxide in

the lung (see Section 5.1.3), and metabolism results in formation of two optically active

enantiomers (R- and S-forms) because of the chiral carbon in styrene-7,8-oxide. Plopper

et al. (1980a, 1980b) identified interspecies morphological differences in Clara cells that

are consistent with the observed differences in styrene metabolism in rodent and human

lung. Mouse and rat Clara cells contain an abundance of agranular endoplasmic

reticulum, which is associated with metabolism of pulmonary toxins via cytochromes

P450. Human Clara cells contain abundant granular endoplasmic reticulum but no

agranular endoplasmic reticulum.

Cohen et al. (2002) reviewed studies measuring conversion of styrene to styrene-7,8-

oxide by cytochrome P450 monooxygenase in pulmonary tissues in rats, mice, and

humans. Most studies showed styrene-7,8-oxide production to be highest in mice (0.95 to

4.5 nmol/min per mg protein), followed by rats (0.32 to 11.7 nmol/min per mg protein),

and humans (0.006 to 0.014 nmol/min per mg protein). Cohen et al. also noted that

metabolic conversion rates varied according to cell type or tissue. Mouse Clara cells

produced styrene-7,8-oxide (193 pmol/106 Clara cells per minute) at 3 times the rate of

rat Clara cells (59 pmol/106 cells per minute); however, when more aggregate pulmonary

tissue fractions (pulmonary microsomes) were compared, the rates differed by a factor of

1.5 (2.13 nmol/min per mg protein in mice vs. 1.44 in rats) (Hynes et al. 1999). However,


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Cohen et al. concluded that differences in styrene-7,8-oxide concentrations in the lung do

not sufficiently explain the differences in susceptibility to the carcinogenic effects of

styrene between rats and mice. Rats did not develop lung tumors in groups that had

similar predicted styrene-7,8-oxide concentrations in the lungs compared with groups of

mice that developed lung tumors.

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Hofmann et al. (2006) investigated the styrene-7,8-oxide levels formed in isolated lungs

of male Sprague-Dawley rats and in-situ prepared lungs from male B6C3F1 mice. Styrene

vapor concentrations were measured in air samples collected in the immediate vicinity of

the trachea and were almost constant during each experiment. Styrene vapor

concentrations ranged from 50 to 980 ppm for rats and 40 to 410 ppm for mice. Both

species metabolized styrene to styrene-7,8-oxide; however, mean styrene-7,8-oxide levels

in mouse lungs were about 2 times higher than in rat lungs at equal exposure conditions.

5.3.2 Detoxification of styrene-7,8-oxide in respiratory tissue 13 Styrene-7,8-oxide is detoxified through hydrolysis mediated by mEH or conjugation with

glutathione mediated by GST. Cohen et al. (2002) summarized studies in rodents and

humans measuring the capacity of mEH in pulmonary tissue to detoxify styrene-7,8-

oxide. The metabolic conversion rates (in nanomoles per minute per milligram of protein)

for hydration of styrene-7,8-oxide by mEH to form styrene glycol varied among studies,

ranging from 0.4 to 2.1 in mice, 0.6 to 2.6 in rats (one study reported < 0.1), and 2.0 to

3.4 in humans. The estimated Kms for hydrolysis from a previous study were 0.013 mM

in mice, 0.0047 mM in rats, and 0.0156 mM in humans. The metabolic conversion rates

for conjugation of styrene-7,8-oxide mediated by GST in pulmonary tissues varied

among studies (as summarized in Cohen et al. 2002); the one study in humans reported a

rate similar to that in one study in mice but lower than the rates from other studies in

mice or rats. The estimated ratio of Vmax to Km (which Cohen stated was an indication of

GST metabolic activity) was much lower in humans (19) compared with mice (171) or

rats (1,982).

Green et al. (2001b) investigated the cytochrome P450-mediated metabolism of styrene

to styrene-7,8-oxide and subsequent metabolism of styrene-7,8-oxide by either mEH or

GST in nasal and liver microsomal fractions from mice, rats, and humans. P450


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metabolism of styrene to styrene-7,8-oxide was similar in rat and mouse olfactory and

respiratory fractions but was not detected in human nasal samples. Rates in rodent

olfactory fractions were higher than those measured in respiratory or liver fractions. The

rates of metabolism of R and S styrene-7,8-oxides via mEH in rat respiratory fractions

were up to 3.5-fold higher while rates in olfactory fractions were up to 10-fold higher

than in mice. Rates of mEH-mediated metabolism of styrene-7,8-oxide in human nasal

fractions were comparable with mouse olfactory and respiratory tissues and rat

respiratory tissues. Rodent nasal and respiratory tissues also metabolized styrene-7,8-

oxide via GST at rates significantly higher than those for mEH. Olfactory fractions from

rats had 3- to 4-fold greater rates of glutathione conjugation than observed in mice. In

contrast, metabolism of styrene-7,8-oxide by glutathione conjugation was undetectable in

5 of 6 samples of human nasal tissues, and the sixth sample metabolized styrene-7,8-

oxide at a much lower rate than did mouse or rat tissues.

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5.3.3 Stereochemistry considerations 14 The metabolic activation of styrene to styrene-7,8-oxide enantiomers has been reported to

depend on tissue and species, and some authors have suggested that the R-enantiomer is

more toxic. Cohen et al. (2002) summarized the results of studies evaluating the ratio of

the R- to S-enantiomers of styrene-7,8-oxide produced through pulmonary and hepatic

metabolism. The results from these studies showed that mouse lung microsomes

produced greater amounts of the R-enantiomer than did microsomes from rat or human

lung. In mouse lung, the R/S ratio was usually between 2.4 and 2.6, although one study

reported a ratio of 1.7; in rat lung, the ratio was 0.52, based on one available study, and in

human lung, it was 1.15, based on 1 sample from one study. In hepatic microsomes, the

R/S ratio ranged from 1.18 to 1.78 in mice and was 0.57 in rats (one study) and 0.72 in

humans (one study). Cohen et al. reported that their PBPK model predicted that the R/S

ratio in mouse and rat lungs would be approximately twice as high as the ratio for total

styrene-7,8-oxide between the two species; nevertheless, these differences were

insufficient to explain the differences in susceptibility. In studies in mice, the R/S ratio

was 4.0 in isolated Clara cells, but 3.6 in type II cells. In rats, on the other hand, Clara

cells produced a nearly racemic mixture of enantiomers, and the S-isomer predominated


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in type II cells. Green et al. (2001b) reported an R/S ratio of approximately 3 in nasal

tissue of rats and mice and liver tissue of mice, and a ratio of 0.72 in liver tissue of rats.

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Linhart (2001) reported that human liver microsomes produced a nearly racemic mixture

of enantiomers; however, 2 samples showed a predominance of the S-isomer. Wenker et

al. (2001b) reported variable enantioselectivity in human liver microsomes, which

produced a moderate excess of the S-isomer at a low styrene concentration (16 μM)

(mean ± SD = 14.7% ± 6.9%) but an excess of the R-isomer at a high styrene

concentration (1,100 μM) (7.0% ± 8.9%). When Wenker et al. (2000) compared the

metabolism of R- and S-styrene-7,8-oxide by 20 human liver microsomal preparations,

they found among the samples a 3- to 5-fold variation in the Vmax, Km, and Vmax/Km values

for the two enantiomers. They were able to demonstrate that the mEH-mediated

hydrolysis of styrene-7,8-oxide favored hydrolysis of the reportedly more toxic R-

enantiomer, because the S-isomer had a higher Km (by a factor of ~6) and Vmax (by a

factor of ~5) than the R-isomer. The authors found no association between enzyme

kinetics and mEH polymorphisms at exons 3 and 4.

Because of the differences in enantiomeric excess found for each metabolite, Wenker et

al. (2001a) concluded that the individual enzymes responsible for the biotransformation

and excretion of styrene-7,8-oxide differed in their enantiomeric selectivity and/or

specificity. Hallier et al. (1995) determined that the R/S ratio ranged from 0.7 to 2.2 in 20

male German workers in the polyester industry exposed to styrene by inhalation at

concentrations ranging from 29 to 41 ppm; the differences in the R/S ratio could not be

explained by differences in individual exposure or in urinary metabolite concentrations.

The authors proposed that interindividual differences in metabolism of styrene to R- and

S-enantiomers were likely related to enzyme polymorphisms. Drummond et al. (1989)

measured excretion of R- and S-enantiomers of mandelic acid in three workers

occupationally exposed to 8-hour time-weighted average styrene concentrations of up to

420 mg/m3 [100 ppm]. The R/S ratios for the three individuals were 1.16, 1.27, and 1.14.

Linhart (2001) reviewed the stereochemistry of styrene biotransformation and concluded

that the ratio of the enantiomers in a target tissue or cell will depend on both the


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stereoselectivity of the formation of styrene-7,8-oxide and the stereoselectivity of the

metabolism of styrene-7,8-oxide. In rats, the formation reaction favors the S-enantiomer,

and detoxification of the R-enantiomer is faster. The formation reaction in mouse liver

and lungs favors the R-enantiomer, but detoxification moves the ratio closer to a racemic

mixture. Linhart concluded that the stereochemistry of styrene biotransformation might

contribute to species differences in toxicity between mice and rats but that it could not be

interpreted as a crucial factor. In addition, the author concluded that the relationship of

styrene stereochemistry to toxic effects in humans could not be interpreted, because

relevant data were lacking.

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5.3.4 Kinetics of styrene metabolism and toxicokinetic models 10 In addition to the cell-specific metabolism of styrene discussed above, several studies

have focused on the kinetics of styrene and styrene-7,8-oxide metabolism in the whole

animal for several species (IARC 2002). In one study, the rate of metabolism of styrene

to styrene-7,8-oxide was compared among species: the order was guinea-pig > rabbit >

mouse > rat. However, for metabolism of styrene-7,8-oxide to styrene glycol by mEH,

the order was rat > rabbit > guinea-pig > mouse. In another study, the rate depended on

the styrene concentration, decreasing from mouse to rat to human at a low concentration,

but from rat to mouse to human at a high concentration (IARC 2002). Cruzan et al.

(2001, 1998) reported that styrene-7,8-oxide concentration in the blood was lower in

mice exposed to a concentration of 160 ppm [an exposure level associated with lung

cancer] than in rats exposed at 1,000 ppm [an exposure level at which no tumors were

observed].

Several pharmacokinetic models have been developed that compared styrene distribution

and metabolism in mice, rats, and humans. Sarangapani et al. (2002) reported that the

earlier models (Csanady et al. 1994, Ramsey and Andersen 1984) did not treat the

respiratory tract as a target organ and did not incorporate metabolic production and

clearance of styrene-7,8-oxide in the respiratory tract. Therefore, the Sarangapani et al.

PBPK model incorporated a multicompartmental description of the respiratory tract and

specifically added a compartment to represent the terminal bronchiolar region. This

model was based on metabolism of styrene in the liver and the terminal bronchiolar


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region of the lung, which is richest in the metabolically active Clara cells and in which

the authors considered styrene-mediated toxicity to occur.

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Filser et al. (2002) also developed a PBPK model for styrene in mice, rats, and humans

based on metabolism in both the liver and lung. This model divided the lung into two

compartments: the gas conducting zone and the gas exchange zone. The enzymatic

capacities of the two compartments were based on their shares of the total lung volume

because the kinetics of styrene and styrene-7,8-oxide metabolizing enzymes were

determined in microsomes and cytosol from whole lung tissue. They tested the validity of

their model by comparing the predicted area under the curve for blood styrene-7,8-oxide

concentration with reported values from published studies in Sprague-Dawley and F344

rats and B6C3F1 mice.

Both of these models predicted that the order of styrene concentration in the lung (Filser

et al.) or terminal bronchioles of the lung (Sarangapani et al.) would be mouse > rat >

human. The Harvard Center for Risk Analysis also developed a PBPK model that

predicted the concentrations of styrene-7,8-oxide and R-styrene-7,8-oxide in the tissues

of humans, rats, and mice exposed to styrene at different concentrations (Cohen et al.

2002). This model used Csanády et al. (1994) as a starting point but included several

modifications (e.g., equations to account for styrene metabolism in the lung and to

estimate R- and S-styrene-7,8-oxide concentrations in various tissues). Results from this

model were inconclusive because of inconsistencies among studies in the measured levels

of styrene-7,8-oxide in the blood. Depending on the data used for calibration, the model

sometimes predicted higher concentrations of styrene-7,8-oxide in the lungs of rats, while

in other cases, higher concentrations were predicted for the mouse. However, Csanády et

al. (2003) reported that there was a typographical error in an equation described in

Csanády et al. 1994 that was overlooked by Cohen et al. (2002) and could explain their

inability to copy the Csanády et al. model.

Although the available data suggested that the mouse has a greater metabolic capacity for

converting styrene to styrene-7,8-oxide, and a greater pharmacokinetic response

(particularly with respect to lung tumors), the data were insufficient to explain why mice


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were more susceptible than rats. These authors concluded that the existing

pharmacokinetic data failed to explain the observed differences in metabolite levels in

mice, rats, and humans. These authors further noted that the current inability to explain

species differences makes it difficult to determine whether the rat or the mouse is the

better model for the human response to styrene.

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5.4 Genetic and related effects 6 Biotransformation of styrene to the genotoxic styrene-7,8-oxide seems to be responsible

for the majority of the genotoxic effects associated with styrene exposure (Cohen et al.

2002). Furthermore, during the manufacture of reinforced plastics, styrene and trace

amounts of styrene-7,8-oxide are released, thus, direct occupational exposure of workers

to styrene-7,8-oxide has been shown (see Section 2.5.1.6) (Nylander-French et al. 1999,

Rappaport et al. 1996, Tornero-Velez and Rappaport 2001). This section summarizes the

publicly-available peer-reviewed literature on the genetic and related effects of styrene.

Styrene genotoxicity has been investigated in many in vitro and in vivo studies and

reviewed in several publications (Barale 1991, Cohen et al. 2002, IARC 1994a, 2002,

Scott and Preston 1994a, Speit and Henderson 2005, Vodicka et al. 2006b). The genetic

and related effects discussed below include studies of DNA adducts, alkali-labile lesions,

DNA strand breaks, cytogenetic damage, and mutations, with a focus on mammalian

systems, especially human cells and studies of styrene-exposed workers.

5.4.1 DNA adduct formation 20 This section discusses formation and chemistry of styrene-7,8-oxide DNA adducts.

Specific studies of DNA adduct formation in cell cultures, experimental animals, and

styrene-exposed workers are discussed in the following sections.

DNA adduct detection methods are important tools for determining the etiology of human

cancer and for measuring metabolic enzyme and DNA repair system genotypes (Collins

1998, Hemminki et al. 2000, Perera and Weinstein 2000). The major metabolite of

styrene in vivo is styrene-7,8-oxide, which is expected to bind covalently to biological

macromolecules. The binding of styrene-7,8-oxide to nucleic acid constituents has been

studied extensively during the last 20 years; however, no studies on other styrene

metabolites with the potential to bind DNA (e.g., styrene 3,4-oxide) were identified.


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Styrene-7,8-oxide possesses two sites (the α- and β-carbons of the epoxide moiety; see

Figure 1-2) that are electrophilic and able to react at nucleophilic sites in DNA. Either

carbon in the epoxide of styrene-7,8-oxide can react with nucleic acid, and because the

carbon atom at the 1-position is a chiral center, there are four possible diastereomers (R-

and S-isomers of the alpha form and R- and S- isomers of the beta form) (Phillips and

Farmer 1994).

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The reaction mechanisms of styrene-7,8-oxide alkylation have been intensively studied

(Barlow and Dipple 1998, 1999, Latif et al. 1988, Qian and Dipple 1995). In this

document, binding sites for adducts are identified by the position of the atom as part of a

ring (the atom to which the adduct is bound is followed by its position in the ring [e.g.,

N3 of deoxyguanosine]) or as an exocyclic group on the ring (the atom to which the

adduct is bound is followed by the position of the ring atom to which it is bound,

superscripted [e.g., N2 of deoxyguanosine]). The primary target of styrene-7,8-oxide

alkylation in DNA is a guanine residue (Hemminki and Hesso 1984, Koskinen et al.

2000b, Koskinen et al. 2000a, Latif et al. 1988, Savela et al. 1986). Styrene-7,8-oxide

forms adducts at the N7-, N2-, and O6-positions of guanine, the N1-, N3-, and N6-

positions of adenine, the N3-, N4-, and O2-positions of cytosine, and the N3-position of

thymine (Figure 5-3). In vitro studies indicated that N7- and N2-alkylguanine and O6-

adducts of guanine adducts were the most abundant, followed by adducts with

deoxycytidine (N3, N4, and O2), deoxyadenosine (N1, N3, and N6), and thymidine (N3)

(IARC 2002). The relative reactivity of the nucleosides with styrene-7,8-oxide are dG >

dC > dA > T, while the alkylation rates of guanine by styrene-7,8-oxide are

deoxyguanosine > single-stranded DNA > double-stranded DNA (Phillips and Farmer

1994, Savela et al. 1986). Vodička and Hemminki (1988) reacted radioactive styrene-7,8-

oxide with double- and single-stranded DNA. The N7-, N2-, and O6-guanine adducts

accounted for at least 95% of the total in single stranded DNA and formed in the

proportions 54:33:12. The proportions were 74:23:3.7 in double-stranded DNA,

indicating suppression at atoms that take part in hydrogen bonding in double-stranded

DNA (N2 and O6). At neutral pH, styrene-7,8-oxide in solution with guanosine alkylated

the nucleoside mainly at the N7-position (57% of identified products), followed by the


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N2- (28%) and O6-positions (15%) (Hemminki and Hesso 1984). When styrene-7,8-oxide

was incubated in vitro with double-stranded DNA, the α and β forms of the N7-guanine

adduct together constituted up to 74% of total adducts formed, while the α form of the

N2-guanine adduct constituted about 3% and the O6-guanine adduct about 1% (Koskinen

et al. 2001b, Vodicka et al. 2002a). The exact proportion of O6-guanine adducts has been

difficult to determine because of their chemical instability; however, the half-life of the α

isomer of O6-guanine adducts in double-stranded DNA has been estimated to be 1,320

hours. Thymidine is a poor substrate for styrene-7,8-oxide, with only minor alkylation

occurring at pH 7.4 and 37°C at the N3-position (Koskinen et al. 1999).

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Figure 5-3. Styrene-7,8-oxide binding sites in DNA (from Vodicka et al. 2002a) Styrene-7,8-oxide-binding sites are indicated by arrows; base-pairing sites in DNA are labeled with

asterisks.

A number of interconversions may occur with styrene-7,8-oxide nucleotide adducts. N1-

adenine adducts can deaminate to form the corresponding hypoxanthine adduct, and N3-

deoxycytidine adducts are rapidly deaminated to the corresponding deoxyuridine. N1-

adenine adducts also may undergo rearrangement to the N6-adduct via the Dimroth

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rearrangement, in which the adducted molecule moves from the ring nitrogen atom to the

exocyclic nitrogen atom (Barlow et al. 1998). O2-cytosine adducts also are unstable,

being prone to depyrimidation and interconversion between the α- and β-isomers

(Koskinen et al. 2000b). The chemically stable DNA adducts identified in vitro in the

highest proportions are α-N6–adenine, α-N2–guanine, and β-N3–uracil (Koskinen et al.

2001b); however, these adducts have not yet been identified in vivo in experimental

animals (Vodicka et al. 2006b). O6-guanine adducts account for about 1% of total

adducts (Vodicka et al. 1994). In the case of O6-guanine adducts, the α-isomer can

convert to the β-isomer in a base-catalyzed rearrangement (Moschel et al. 1986).

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Structural data for both α- and β-N6–dA adducts of styrene-7,8-oxide in DNA is

available. The α-N6–dA adducts locate in the major groove, with their orientation being

dependent upon stereochemistry. Adducts with R-stereochemistry orient in the 5'

direction; whereas, those with S-stereochemistry orient in the 3' direction (Feng et al.

1995, 1996, Stone and Feng 1996). While the adducts with S-tereochemistry induce a

slight bend in the duplex, those with R-stereochemistry do not (Le et al. 2000). The

structures of the diastereomeric α-N6–dA adducts mispaired with dC, have also been

examined, both in the 5'-CXA-3' sequence and the 5'-AXG-3' sequence. This represents

the putative intermediate leading to A to G transitions. In the former sequence, the adduct

with S-stereochemistry remains in the major groove and oriented in the 3'-direction, as

observed for the corresponding adduct paired correctly with thymine. A shift of the

modified adenine toward the minor groove results in the styrenyl ring stacking with the

5'-neighboring cytosine, which shifts toward the major groove. A wobble A•C base pair

is not observed. In this mismatched duplex, the adduct of R-stereochemistry is disordered

(Painter et al. 1999). In the 5'-CXA-3' sequence, the thermodynamic stability of both the

mismatched R- and S-adducts is dependent upon pH. At neutral pH, both exhibit

significant structural perturbations and lower Tm values, as compared with the 5'-CXA-3'

sequence. This is attributed to reorientation about the adenine C6-N6 bond. For the adduct

of R-stereochemistry, the styrenyl moiety remains oriented in the major groove but now

orients in the 3'-direction. For the adduct with S-stereochemistry, the styrene ring inserted

into the duplex, approximately perpendicular to the helical axis of the DNA, but now in


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the 5'-direction (Simeonov et al. 2000). The increased tether length of the β-styrene-7,8-

oxide N6-dA adducts results in two changes in structure as compared with the α-styrene

adducts. First, less distortion is introduced into the duplex. For both the R- and S-β-N6-

dA adducts, the styrenyl moiety is accommodated within the major groove of the duplex

with little steric hindrance. Second, it mutes the influence of stereochemistry, such that in

contrast to the α-N6-dA adducts, either the R- or S-stereoisomeric β-N6-dA adducts

exhibit similar conformations within the major groove (Hennard et al. 2001).

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5.4.2 In vitro studies 8 This section reviews in vitro studies of DNA adduct formation (5.4.2.1), DNA damage

and repair (5.4.2.2), mutagenicity (5.4.2.3), and cytogenetic markers (5.4.2.4) for styrene

and styrene-7,8-oxide.

5.4.2.1 DNA adducts 12 The various types of DNA adducts associated with exposure to styrene or styrene-7,8-

oxide (based on the binding site on the nucleotide) are shown in Figure 5-3.

Styrene 15 No in vitro DNA adduct studies with styrene were identified.

Styrene-7,8-oxide 17 IARC (1994b, 2002) and Philips and Farmer (1994) reviewed several in vitro studies on

DNA adduct formation in nucleosides, calf thymus or fish testis DNA, and in DNA in

mammalian and human cells exposed to styrene-7,8-oxide. Studies in cellular systems are

summarized in Table 5-2. Exposure of 9L cells [rat brain gliosarcoma cells] to 1 mM

styrene-7,8-oxide, [a concentration that has resulted in increased chromosome aberrations

in human lymphocytes in vitro], for 24 hours resulted in formation of several DNA

adducts (Liu et al. 1988a). DNA adduct formation in human cells exposed to styrene-7,8-

oxide has been studied in cultured peripheral blood lymphocytes (PBLs), human

embryonic lung fibroblasts (HEL), and keratinocytes. DNA adducts (O6-guanine, N7-

guanine, or N2-guanine) were induced in all cell lines. These studies indicated a dose-

related increase in DNA adducts, persistence of the O6-guanine adducts, and a correlation

with single-strand breaks (see Section 5.4.2.2). In HEL cells, lower levels of N7-guanine


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adducts were observed after 18-hour than 3-hour exposures; this finding could be due to

the conversion of N7-guanine adducts into abasic sites either spontaneously or through

the DNA repair process (Vodicka et al. 1996). Pauwels and Veulemans (1998) also

reported N7-guanine adducts of styrene-7,8-oxide with human DNA when whole blood

was incubated with styrene-7,8-oxide at 9.4 to 460 mM; however, they did not report the

numbers of adducts formed.

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Adduct persistence varies by binding site, and the time of exposure to the alkylating

agent largely determines the relative proportions of the various DNA adducts. The

available data indicate that the N7-adducts are lost, with an estimated half-life of about

19 hours in one study (Vodicka et al. 1996). Some data indicate that O6 adducts are stable

and can build up over time from chronic low-level exposures (Bastlová et al. 1995,

Vodicka et al. 1999, Vodicka et al. 1994). An apparent saturation level is reached

considerably faster for the N7-guanine and N3-adenine adducts, because these

nucleotides depurinate more readily than adducts formed at positions involved in base-

pairing (see Figure 5-3) (Vodicka et al. 2002a).

Table 5-2. Styrene-7,8-oxide DNA adducts formed in mammalian cells in vitro Treatment Adducts

Cell type Conc. (μM) Duration Type No. per 108 dNp Reference Rat 9L (gliosarcoma cells) 1,000 24 h NR 1,950 Liu et al.

1988a

Human whole blood

9,400 49,000 96,000

240,000 460,000

2 h N7-guanine NR Pauwels and Veulemans 1998

200 400 600

24 h O6-guanine 0.98–2.04

2.46 2.59–4.05

200 6 d O6-guanine 1.2

Bastlová et al. 1995 Human

lymphocytes

600 24 h N7-guanine N2-guanine

580 7

Vodicka et al. 2002a

10 50

100 3 h N7-guanine

10a 20a 35a Human embryonic

lung fibroblasts 10 50

100 18 h N7-guanine

4a 8a 9a

Vodicka et al. 1996

Human keratinocytes

100 300 NR N7-guanine 140

470 Vodicka et al. 2002a


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Treatment Adducts

Cell type Conc. (μM) Duration Type No. per 108 dNp Reference 100 300 NR N2-guanine 0.8

1.4 dNp = dinucleotide pair; NR = not reported. a Levels were estimated from figures.

5.4.2.2 DNA damage and repair 1 Assays for DNA damage may detect double-strand and single-strand breaks, alkali-labile

sites, oxidative DNA base damage, or crosslinks (DNA-DNA or DNA-protein). In

addition, base and nucleotide excision repair processes also induce transient breaks;

therefore, a high level of breaks may indicate high levels of DNA damage or repair

(Collins et al. 1997). Collins et al. noted that single-strand breaks are quickly repaired

and are not generally regarded as significantly lethal or mutagenic lesions. Genotoxic

agents may directly induce single-strand breaks or may create apurinic/apyrimidinic sites,

which are converted to strand breaks in the alkaline electrophoresis solution used in the

comet assay.

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Styrene 11 Two studies in Escherichia coli strain PQ37 gave somewhat conflicting results for DNA

repair as measured by the SOS chromotest. One study gave negative results, and the other

was inconclusive, showing positive results but no dose-response relationship (IARC

1994a)

Only one study of single-strand breaks was identified. Sina et al. (1983) developed an

alkaline elution/rat hepatocyte assay to measure DNA single-strand breaks and tested the

method on 91 compounds, including styrene and styrene-7,8-oxide. Rat hepatocytes were

treated with styrene at concentrations of 0.03, 0.3, and 3 mM for 3 hours. Single-strand

breaks were significantly increased at the highest concentration compared with controls.

Styrene-7,8-oxide 21 IARC (1994b) reviewed four studies of SOS induction in bacteria (one in S. typhimurium

and three in E. coli). Three studies resulted in a positive response without metabolic

activation. One study in E. coli was negative with or without metabolic activation.


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Styrene-7,8-oxide induced single-strand breaks and DNA damage in rat hepatocytes

(Sina et al. 1983), neuroadrenergic (pheochromocytoma) rat PC-12 cells (Dypbukt et al.

1992), human PBLs and HEL cells (Bastlová et al. 1995, Laffon et al. 2001b, 2002b,

Laffon et al. 2003b, Vodicka et al. 1996), rat and human testicular cells (Bjørge et al.

1996), and Chinese hamster V79 lung fibroblast cells (Herrero et al. 1997). The results

are summarized in Table 5-3. These studies indicated that single-strand breaks increased

in a dose-related manner and were correlated with formation of DNA adducts.

Additionally, Bastlová et al. (1995) and Vodicka et al. (1996) showed that single-strand

breaks in DNA in human PBLs and HEL cells were repaired rapidly, with approximate

half-lives of 40 to 80 minutes. Vodicka et al. (1996) concluded that N7-guanine adducts

were important in the formation of single-strand breaks, because of their strong

correlation in HEL cells. Higher concentrations of styrene-7,8-oxide were required to

induce single-strand breaks in Chinese V79 hamster cells engineered to express human

mEH than in cells lacking this enzyme, suggesting that mEH might have protective

effects (Herrero et al. 1997) (see Sections 5.1.3.6 and 5.3.2 regarding the role of mEH in

detoxification). Marczynski et al. (1997b) exposed human whole blood to styrene-7,8-

oxide for 1.5 to 4 hours and reported that the observed degradation of high molecular

weight-DNA fragments in white blood cells was likely due to oxidative stress.

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Table 5-3. DNA damage in mammalian cells exposed to styrene-7,8-oxide

Cell type

Treatment concentration

(time) Assay method LEC/HIC Results Reference

Rat hepatocytes 30–3,000 μM (3 h)

Alkaline filter elution 300 μM + Sina et al.

1983

Rat PC 12 (neuroadrenergic cells)

30–1,000 μM (1 h)

Alkaline filter elution 30 μM

+ Repair after 3 h in fresh medium: 30 µM = 100%, 100

μM = 40% No double-strand breaks or cross-

links

Dypbukt et al. 1992

Chinese hamster V79 (lung fibroblast cells)

10–1,000 μM (1 h)

Alkaline filter elution

50 μM (mock-transfected cells) 200 μM (hmEH-transfected cells)

+ Herrero et al. 1997

10–100 μM (1 h) Comet assay

Concentration-dependent increase

+ Levels reduced

after 1-2 h in fresh medium and

restored to control levels after 24 h

Bastlová et al. 1995

0.06–0.18 μmola (1.5–4 h)

Pulsed-field and conventional gel electrophoresis

0.06 μmola

+ No clear

association with length of exposure

Marczynski et al. 1997b

10–200 μM (0.5 h) Comet assay 50 μM

+ DNA damage was correlated with SO

concentration

Laffon et al. 2001b


+ Levels reduced after 30 min in

fresh medium and restored to control levels (high-dose group) after 4 h

Laffon et al. 2002b

Human lymphocytes


+ Increased damage

was associated with decreasing

EH activity

Laffon et al. 2003b

Human embryonic lung fibroblasts

10–100 μM (3–18 h)

Alkaline DNA unwinding and separation of ds and ss DNA by hydroxyapatite

chromatography

Concentration-dependent

increase at 3 h(P = 0.003)

+ Levels were ~3-fold higher after treatment for 3 h

compared with 18 h at 100 μM

Vodicka et al. 1996

Human and rat testicular cells

10–300 μM (0.5 h)

Alkaline filter elution 100 μM + Bjørge et al.

1996 ds = double-stranded; hmEH = human microsomal epoxide hydrolase; HIC = highest ineffective concentration; LEC = lowest effective concentration; SO = styrene-7,8-oxide, ss = single-stranded; SSB = single-strand breaks. aReported by Marczynski et al. (1997b) as dose in μmol.

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5.4.2.3 Mutagenicity 1 The mutagenicity of styrene and styrene-7,8-oxide has been investigated in a number of

in vitro systems and is discussed below and summarized in Table 5-4.

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Styrene 4 Most of the studies on styrene mutagenicity in bacterial systems were conducted two or

three decades ago, and the results were reviewed by IARC (1994a, 2002). Briefly, in

Salmonella typhimurium strains, the majority of studies on reverse mutation gave

negative results without metabolic activation. A few studies reported positive results with

metabolic activation in TA100, TA1530, and TA1535, which detect base-pair

substitutions. In eukaryotes, positive results were reported for Saccharomyces cerevisiae

(reverse mutation and gene conversion), Drosophila melanogaster (sex-linked recessive

mutation in one study), and hprt mutations in Chinese hamster V79 cells with metabolic

activation (one study). Negative results were reported in two studies of forward mutations

in Schizosaccharomyces pombe, one study of w/w+ somatic mutations in D.

melanogaster, and two studies of hprt mutations in Chinese hamster V79 cells without

metabolic activation (IARC 1994a, 2002).

Styrene-7,8-oxide 17 Styrene-7,8-oxide was mutagenic in the majority of in vitro systems, primarily without

metabolic activation. Positive results were found in S. typhimurium, E. coli (SOS

chromotest), Klebsiella pneumoniae, S. cerevisiae, S. pombe, and D. melanogaster

(mixed results) and at the tk locus in mouse lymphocytes, the hprt locus in Chinese

hamster V79 cells, and the HPRT locus in human T lymphocytes and B lymphoblastoid

cells (weakly positive). Negative results were found in the D. melanogaster w/w+

somatic mutation assay (Rodriguez-Arnaiz 1998). Bastlová and Podlutsky (1996)

characterized HRPT mutations induced by styrene-7,8-oxide in T lymphocytes. They

found that the dominating base substitution in the HPRT gene was an A·T→G·C

transition, followed by G·C→T·A and A·T→T·A transversions. The DNA adducts

resulting in some of these base substitutions were tentatively identified as N6-

alkyladenine (A·T→G·C transition) and N7-alkylguanine (G·C→T·A and A·T→T·A

transversions).


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Several studies also have compared the mutagenicity of styrene-7,8-oxide enantiomers

(Pagano et al. 1982, Seiler 1990, Sinsheimer et al. 1993). Pagano et al. (1982)

investigated the mutagenic properties of the R-enantiomer, the S-enantiomer, and a

racemic mixture of R- and S-enantiomers in S. typhimurium TA100. The order of

mutagenicity was R-enantiomer > racemic mixture > S-enantiomer. Seiler (1990)

reported on similar studies with styrene-7,8-oxide enantiomers in S. typhimurium TA100;

an intrinsic difference in the mutagenic activity of the enantiomers was strongly

suggested by evidence of qualitative differences in their binding to DNA. Sinsheimer et

al. (1993) also found the R-enantiomer to be a more potent mutagen in S. typhimurium

than the S-enantiomer; however, these results were not predictive of in vivo genotoxicity

in mice where the S-enantiomer rather than the R-enantiomer was associated with an

increase in chromosomal aberrations and sister chromatid exchange (SCE).

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Table 5-4. Mutagenicity of styrene and styrene-7,8-oxide in vitro Styrene Styrene-7,8-oxide

Test system –S9 +S9 –S9 +S9 S. typhimurium (reverse mutation) – ± + ± E. coli (SOS chromotest) –a NT + NT K. pneumoniae (forward mutation) NT NT + NT S. cerevisiae (reverse mutation & gene conversion) + NT +b NT S. pombe (forward mutation) – – + NT D. melanogaster (sex-linked recessive) + NT + NT D. melanogaster (somatic w/w+) –c NT NT NT Chinese hamster V79 cells (hprt) – + + – mouse lymphoma L5178Y (tk) NT NT + – human lymphocytes (HPRT) NT NT (+) NT human T lymphocytes (HPRT) NT NT + NT human B lymphoblastoid cells (HPRT) NT NT (+) NT Source: IARC 1994a, 1994b, 2002. + = positive results or generally positive results in multiple studies; (+) = weakly positive results; ± = mixed results; – = negative results or generally negative results in multiple studies; hprt = hypoxanthine phosphoribosyl transferase gene (mouse); HPRT = hypoxanthine phosphoribosyl transferase gene (human); NT = not tested; tk = thymidine kinase gene (mouse). aNegative or inconclusive results in the SOS chromotest for DNA repair. bPositive results for gene conversion only. cA positive result was reported by IARC (2002) for insecticide-resistant strains, which have high bioactivation capacities.


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5.4.2.4 Cytogenetic markers 1 The cytogenetic effects of styrene and styrene-7,8-oxide have been extensively reviewed

(Barale 1991, Cohen et al. 2002, IARC 1994a, 1994b, 2002, Scott and Preston 1994b)

and are summarized below. Both styrene and styrene-7,8-oxide cause cytogenetic damage

in various cell types tested in vitro. End points investigated include SCE, chromosomal

aberrations, micronuclei, and aneuploidy.

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Styrene 7 Results of in vitro cytogenetic studies with styrene are summarized in Table 5-5. All

studies with human lymphocytes gave positive results. Scott and Preston (1994a) noted

that chromosomal aberrations and SCEs in human lymphocytes increased in the presence

of erythrocytes (i.e., in whole-blood cultures). Erythrocytes have the capacity to oxidize

styrene to styrene-7,8-oxide (see Section 5.1.3.5), while lymphocytes have the potential

to inactivate styrene-7,8-oxide through metabolism by mEH.

SCE were observed in rat and human lymphocytes and in Chinese hamster ovary (CHO)

cells under certain test conditions (IARC 1994a, 2002, Scott and Preston 1994a). In one

study (de Raat 1978), SCE were induced in CHO cells only when metabolic activation

(S9 fraction) was combined with incubation with cyclohexene oxide, an mEH inhibitor,

suggesting that styrene is metabolically activated to styrene-7,8-oxide but rapidly

inactivated by mEH. In another paper reporting six experiments with CHO cells (Norppa

and Tursi 1984), styrene at high concentrations (8 to 12 mM) caused SCE in one of three

experiments without metabolic activation and in two experiments in the presence of

human erythrocytes, but did not cause SCE in one experiment in the presence of S9. [The

high concentrations of styrene used in these experiments limit the interpretation of these

studies.]

Chromosomal aberrations were reported in studies with Allium cepa root-tip cells and

human lymphocytes exposed to styrene and in two of three studies in Chinese hamster

lung cells (weakly positive results). Micronucleus formation occurred in human

lymphocytes and A. cepa root-tip cells (IARC 1994a, 2002, Scott and Preston 1994a). A

strong c-mitotic effect and disordered anaphases were reported in A. cepa root-tip cells,


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and aneuploidy occurred in human lymphocytes (Linnainmaa et al. 1978a, 1978b) but not

in D. melanogaster (Penttila et al. 1980).

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2


Table 5-5. Cytogenetic effects of styrene in vitro

End point Test system Metabolic activationa

LEC/HIC (mM)b Results References

Human lymphocytes (isolated cultures or whole blood)c

– – – – – –

2.0 0.7 0.5 1.0

0.01 0.5

+ + + + + +

Norppa et al. 1983a Norppa et al. 1980a Norppa et al. 1983a Norppa and Vainio 1983 Chakrabarti et al. 1993 Lee and Norppa 1995

CHO cells – S9

S9 + CO – – –

S9 HE HE

8.7 8.7 4.4 15 12 12 20 8

12

– – + – – + – + +

de Raat 1978 de Raat 1978 de Raat 1978 Norppa and Tursi 1984 Norppa and Tursi 1984 Norppa and Tursi 1984 Norppa and Tursi 1984 Norppa and Tursi 1984 Norppa and Tursi 1984

SCE

Rat lymphocytes (whole blood) – 0.5 + Norppa et al. 1983b

Human lymphocytes (isolated cultures or whole blood)c

– – – –

1.0 2.6 0.5 2.0

+ + + +

Jantunen et al. 1986 Linnainmaa et al. 1978a, 1978b Pohlova and Sram 1985 Jantunen et al. 1986

Chinese hamster lung cells

– S9 –

2.4 2.4 1.0

– (+) (+)

Matsuoka et al. 1979 Matsuoka et al. 1979 Ishidate and Yoshikawa 1980

Chromosomal aberrations

A. cepa – 0.87 + Linnainmaa et al. 1978a, 1978b Human lymphocytes (whole blood)

– 2.6 + Linnainmaa et al. 1978a, 1978b Micronuclei

A. cepa – 1.7 + Linnainmaa et al. 1978a, 1978b Human lymphocytes (whole blood)

– 2.6 + Linnainmaa et al. 1978b Aneuploidy

D. melanogaster – 5 – Penttila et al. 1980 C-mitosis A. cepa – 0.87 + Linnainmaa et al. 1978a, 1978b Source: adapted from Scott and Preston 1994a and IARC 1994a, 1994b, 2002. + = positive response; (+) = weak positive response; – = negative response. a CO = cyclohexene oxide, an inhibitor of epoxide hydrolase; HE = human erythrocytes; S9 = phenobarbital or 3-methylcholanthrene-induced post-mitochondrial supernatant fraction of rat liver homogenate. b Lowest effective concentration or highest ineffective concentration. c Erythrocytes in whole-blood preparations can act as a metabolic activation system (Norppa et al. 1983b) (see Section 5.1.3.4).

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Styrene-7,8-oxide 1 Styrene-7,8-oxide induced cytogenetic effects at lower concentrations than did styrene,

and metabolic activation was not necessary. Results are summarized in Table 5-6. SCEs

occurred in human lymphocytes, CHO cells, and Chinese hamster V79 cells.

Chromosomal aberrations occurred in human lymphocytes and Chinese hamster V79

cells but not in A. cepa. Micronuclei were induced in human lymphocytes, Chinese

hamster V79 cells, and A. cepa. Linnainmaa et al. (1978a, 1978b) also reported anaphase

bridges in A. cepa cells, which induced micronuclei in successive telophases and

interphases. It was not possible to assess incidences of aneuploidy in human lymphocytes

exposed to styrene-7,8-oxide because of severe chromosome destruction.

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5.4.3 In vivo studies in experimental animals 11 5.4.3.1 DNA adducts 12 As mentioned above, styrene does not bind to DNA unless metabolically activated to

styrene-7,8-oxide. The potential of styrene or styrene-7,8-oxide exposure to induce DNA

adducts in experimental animals was studied earlier through the use of radiolabeled

compounds, as reviewed by Phillips and Farmer (1994) and Cohen et al. (2002). These

studies generally showed low levels of DNA adducts in rats and mice following exposure

to styrene or styrene-7,8-oxide by various routes of administration. However, the reported

levels of DNA binding varied by factors of 20 to 50 among studies, for reasons that were

not completely understood. According to Phillips and Farmer, differences in route of

administration, methods of measurement, and losses from depurination should be

considered.


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Table 5-6. Cytogenetic effects of styrene-7,8-oxide in vitro, without metabolic activation

End point Test system LEC/HICa

(mM) Results References Human lymphocytes 0.07

0.15 0.008 0.1

0.05 0.05 0.1

0.05 0.05

+ + + + + + + + +

Norppa et al. 1980a Norppa et al. 1983a Pohlova and Sram 1985 Zhang et al. 1993 Lee and Norppa 1995 Uüskula et al. 1995 Chakrabarti et al. 1997 Ollikainen et al. 1998 Laffon et al. 2001b

CHO cells 0.18 + de Raat 1978

SCE

Chinese hamster V79 cells

0.17 0.12

+ +

Nishi et al. 1984 von der Hude et al. 1991

Human lymphocytes 0.59 0.1 0.2

0.024

+ + + +

Linnainmaa et al. 1978a, 1978b Fabry et al. 1978 Norppa et al. 1981b Pohlova and Sram 1985

Chinese hamster V79 cells 0.75 + Turchi et al. 1981


A. cepa 3.7 – Linnainmaa et al. 1978a, 1978b Human lymphocytes 0.59

0.1 + +

Linnainmaa et al. 1978a, 1978b Laffon et al. 2001b

Chinese hamster V79 cells 0.75 + Turchi et al. 1981

Micronuclei

A. cepa 3.7 + Linnainmaa et al. 1978a, 1978b Anaphase bridges

A. cepa 0.74 + Linnainmaa et al. 1978a, 1978b

Source: adapted from Scott and Preston 1994a; IARC 1994a, 1994b, 2002. + = positive response; – = negative response. aLowest effective concentration or highest ineffective concentration.

Styrene 1 Adducts resulting from exposure to tritiated styrene were detectable in 2 of 4 lung

samples from female rats and in mouse liver but not in rat liver; however, no lung tissue

was collected from mice in this study (Cantoreggi and Lutz 1993). The earlier study by

Byfält-Nordqvist et al. (1985) with 14C-labeled styrene in NMRI mice reported adduct

values 20 to 50 times those reported by Cantoreggi and Lutz. Philips and Farmer (1994)

were not able to identify a reason for the difference although they noted that the methods

differed for route of adimistration (inhalation, ingestion, and i.p. injection) and in the

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quantitation of radioactivity (coelution with adduct standards vs. measurement of total

radioactivity, but they did not consider these differences sufficient to explain the widely

differing results.

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More recent studies have focused on the quantitative and qualitative determination of

specific styrene-induced DNA adducts (Boogaard et al. 2000b, Gamer et al. 2004,

Otteneder et al. 2002, Pauwels et al. 1996, Vodicka et al. 2001b, Vodicka et al. 2006a).

The studies are reviewed below and summarized in Table 5-7.

DNA adducts resulting from exposure to styrene were detected in tissues from male

NMRI mice in several studies by Vodicka and co-workers (Pauwels et al. 1996, Vodicka

et al. 2001b, Vodicka et al. 2006a). In the only study using i.p. injection (Pauwels et al.

1996), styrene was administered at 0 to 4.35 mmol/kg b.w., and tissues were collected 3

hours later. N7- and O6-guanine adducts were present in the lungs, liver, and spleen, but

N7 adducts were more abundant in all three tissues, and the lungs contained

approximately 30% more of these adducts than did the liver or spleen. The authors

pointed out that the liver would be expected to be exposed to styrene as the first-pass

organ, but they suggested that the balance between formation and detoxification of

styrene-7,8-oxide in the organs could explain the higher adduct levels in lung. DNA

adduct levels correlated with exposure level and formation of hemoglobin adducts.

In the inhalation studies, β-N7–guanine adducts were detected in the lungs but not liver,

and β-N1–adenine adducts were detected in both lungs and liver of male NMRI mice

exposed to styrene at 750 or 1,500 mg/m3 [175 or 350 ppm] 6 hours per day, 7 days per

week, for 1, 3, 7, or 21 days (Vodicka et al. 2001b, 2006a). Levels of both types of DNA

adducts in the lungs correlated significantly with styrene concentrations in blood as a

measure of styrene exposure. Levels of N7-guanine adducts were compared between the

lungs and the urine (with correction for depurination); the total N7-guanine adducts (23.0

adducts/108 nucleotides) in the lungs of mice in the highest exposure group (1,500 mg/m3

[350 ppm] for 21 days) accounted for approximately 0.5% of the total N7-guanine

adducts measured by cumulative urinary excretion.


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DNA adducts also were detected in liver, lungs, and isolated lung cells of male CD-1

mice and male Sprague-Dawley rats exposed to [ring-U-14C]styrene at 160 ppm by nose-

only inhalation for 6 hours (Boogaard et al. 2000b). Tissues were collected either

immediately or 42 hours after exposure. Low levels of N7-guanine adducts were detected

in both liver and lung; however, unidentified adducts [authors’ term] were present in liver

at higher levels than the N7-guanine adducts. The level of N7 adducts and of two of the

three unidentified adducts increased from 0 to 42 hours. N7 adducts were the major

adduct type in the lungs, at a level of about 1 per 108 nucleotides immediately after

exposure and at about half this level 42 hours after exposure. Adducts also were

measured in Clara cells and non-Clara cells. Adducts were analyzed in lung tissue from 2

mice and lung cells from 24 mice. N7-guanine adduct levels were similar in Clara cells,

non-Clara cells, and whole lung. After 42 hours, an unidentified adduct was detected at

levels of 6 per 108 nucleotides in Clara cells and at 80 per 108 nucleotides in non-Clara

cells. The authors stated that because of the small amount of DNA isolated from non-

Clara cells, this value had a large relative error (approximately 30%). The authors used

styrene metabolite standards to identify this adduct (which was the same for both lung-

cell types) and found that benzoic acid co-eluted with the compound. They speculated

that the most likely source was benzaldehyde, which is a putative intermediate in the

metabolism of styrene from mandelic acid to hippuric acid; however, they also suggested

that this adduct could be an artifact resulting from the radioactive styrene used for the

exposure.

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N7-guanine adducts were detected in both rat liver and lung; however, the unidentified

adducts that were present in mouse liver at higher levels than the N7-guanine adducts

(see above) were not detected in rat liver (Boogaard et al. 2000b). One rat was used for

the adduct analysis from lung tissue and five rats were used for the cell-type analysis. N7-

adduct levels were approximately 1 adduct per 108 nucleotides immediately after

exposure, and about half this level at 42 hours after exposure. Type II cells isolated from

lungs of styrene-exposed rats contained higher levels of N7-guanine adducts (2 adducts

per 108 nucleotides) than whole lung.


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Otteneder et al. (2002) did not detect O6-guanine adducts in the lungs or liver of CD-1

mice exposed to styrene at 40 or 160 ppm for 2 weeks. Gamer et al. (2004) also reported

that no changes were found in 8-hydroxy-deoxyguanosine as an indicator of oxidative

stress after either a single 6-hour exposure or multiple exposures of female CD-1 mice to

styrene by inhalation; however, they did find that glutathione was depleted in lung

homogenates.

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In CD-1 rats exposed for 2 years to styrene at 1,000 ppm (the highest level tested), both

α-and β-O6–guanine adducts were detectable in the liver at levels of 90 per 108

nucleotides in males and 80 per 108 nucleotides in females (Otteneder et al. 2002). An

isomer-enriched analysis was used for the 1,000-ppm samples only, and all of the lung

tissue was used for histopathological analysis. However, no O6-guanine adducts were

detected in the lungs or liver of CD-1 rats exposed to styrene at 500 ppm for 2 weeks.

Overall, the N7- and O6-guanine adducts were found most often in these studies. As

noted in Section 5.4, the N7-guanine adducts are the most common form resulting from

exposure to styrene, but the O6-guanine adducts are more persistent, which may explain

their detection along with the N7-adducts.

Table 5-7. Formation of styrene-7,8-oxide DNA adducts in animals exposed to styrene

Adducts

Species Exposurea Type No./108

nucleotidesb Reference

N7-guaninec lung: [63.5] liver: [47.6] spleen: [36.7]

Male NMRI mice

0–4.35 mmol/kg b.w measured at 3 h

O6-guaninec lung: [37.8] liver: [24.7] spleen: [25.7]

Pauwels et al. 1996

βN7-guanine lung: 23 liver: ND

Male NMRI mice

[175 or 350 ppm], 6 h, 7 days/wk, for 1, 3, 7, or 21 d

βN1-adenine lung: 0.6 liver: 0.2

Vodicka et al. 2001b Vodicka et al. 2006a


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Adducts

Species Exposurea Type No./108

nucleotidesb Reference

N7-guanine 42 h lung: ~ 0.5 Clara: ~< 1 non-Clara: ~4 liver: 3.2

Male CD-1 mice

160 ppm for 6 h, measured at 0 and 42 h post-exposure

unidentifiedd 42 h lung: < 1.0 Clara: 6 non-Clara: 80e liver: 8–11

Boogaard et al. 2000b

Female CD-1 mice

40 or 160 ppm, 6 h, 5 d/wk for 2 wk

O6-guanine lung: < detection limit of 1–5/107)

Otteneder et al. 2002

Female CD-1 mice

40 or 160 ppm, 6 h: single exposure or 5 or 20 d

8-OH-deoxyguanosine

lungs: no evidence of oxidative stress

Gamer et al. 2004

N7-guanine 42 h lung: ~0.5 type II cells: 2 non-type II cells: NRf liver: 1.9

Male Sprague-Dawley rats

160 ppm, 6 h, measured at 0 and 42 h post-exposure

unidentified lung: < 0.5 liver: < 0.5

Boogaard et al. 2000b

Female CD rats

500 ppm (rats), 6 h/d for 2 wk O6-guanine lung: < detection limit of 1–2/107


Male and female CD rats

1,000 ppm, 6 h, 5 d/wk for 2 yr O6-guanine liver: 90 (males) 80 (females)


Female Crl:CD rats

40 or 160 ppm, 6 h: single exposure or 5 d

8-OH-deoxyguanosine

lung: no evidence of oxidative stress

Gamer et al. 2004

a Styrene exposure was by inhalation in all studies except that of Pauwels et al., which used i.p. injection. b Adduct levels are the highest reported for each study unless otherwise indicated. c Adduct levels were converted from femtomoles per milligram of DNA based on the assumption that 1 mg DNA = 3.24 μmol of nucleotides. d Not the same adduct for all values; in the liver, the peaks eluted at 9 and 37 minutes, and in the lung cells, the major peak was at 9 minutes. e Based on only 170 μg of DNA; the authors stated that the error may be approximately 30%. f N7 adducts were present in non-type II cells immediately after exposure, but the concentration of adducts at 42 hours could not be accurately determined because of the low yield of DNA.

Styrene-7,8-oxide 1 Philips and Farmer (1994) reported that very low levels of DNA adducts were formed in

the forestomach [the target tissue for styrene-7,8-oxide–induced tumors] and liver when

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tritiated styrene-7,8-oxide was administered by gavage to rats and by i.p. injection to

mice (Cantoreggi and Lutz 1992, Lutz et al. 1993). An earlier report by Byfält-Nordqvist

et al. (1985) in which tritiated styrene-7,8-oxide or styrene was administered by i.p.

injection to NMRI mice reported that alkylation of DNA in liver, brain, and lung

exceeded that in spleen and testis, but the forestomach was not examined.

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5.4.3.2 DNA damage and repair 6 Results from studies of DNA damage in experimental animals exposed to styrene or

styrene-7,8-oxide are summarized in Table 5-8 and discussed below. Most of the studies

used the alkaline single-cell gel electrophoresis (comet) assay. The comet assay involves

embedding single cells in agarose gel followed by lysis in alkali and electrophoresis

(Vaghef and Hellman 1998). This assay can detect strand breaks, alkali-labile sites

(converted to single-strand breaks under the alkaline conditions of the assay), oxidative

base damage, crosslinks, and DNA repair.

Styrene 14 DNA damage was observed in two studies in mice exposed to styrene by i.p. injection

(Vaghef and Hellman 1998, Walles and Orsen 1983). Walles and Orsen (1983)

administered styrene at 1.7 to 10.1 mmol/kg b.w. [177 to 1,052 mg/kg b.w.] to male

NMRI mice and determined DNA damage in kidney, liver, lung, testis, and brain at 1 to

24 hours after injection. DNA damage was increased in all tissues examined at 1 hour,

and the levels were still elevated at 24 hours in all tissues but the liver. Vaghef and

Hellman (1998) determined DNA damage in peripheral blood lymphocytes, bone

marrow, liver, and kidney cells at 4 and 16 hours after i.p. injection of 100 to 500 mg/kg

b.w. of styrene to female C57BL/6 mice. Significant increases in DNA damage were

found in all tissues at both 4 and 16 hours.

Inhalation studies are usually conducted over a long period, to ensure that equilibrium

between DNA damage and repair is reached; however, only subacute inhalation studies

of styrene have been conducted. Vodicka et al. (2001b) exposed male NMRI mice to

styrene by inhalation at a concentration of 750 or 1,500 mg/m3 [175 or 350 ppm] for 7 or

21 days; they found significant increases in DNA damage only in lymphocytes at 7 days,

and not in bone marrow or liver cells. Endonuclease III–sensitive sites in bone marrow


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were increased significantly at 21 days at both exposure levels, suggesting an increase in

accumulation of abasic sites.

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Only one study that examined DNA damage in styrene-exposed rats was identified.

Kligerman et al. (1993) exposed female F344 rats to styrene by inhalation at 125, 250, or

500 ppm, 6 hours per day for 14 consecutive days. No significant increase in DNA

damage was detected.

Clay (2004) reported that styrene induced DNA damage and repair in female CD-1 mice

in an assay for unscheduled DNA synthesis (UDS). Groups of 6 mice were exposed to

styrene by inhalation at either 125 or 250 ppm for 6 hours. A positive control group was

administered N-nitrosodimethylamine (NDMA) at 10 mg/kg b.w. by gavage. Groups of 3

mice were killed for tissue collection at 2 and 16 hours after exposure to styrene or

NDMA. No increase in UDS was observed for any of the animals exposed to styrene, but

the positive control induced increases in UDS that the author characterized as

appropriate.

Styrene-7,8-oxide 15 After a single i.p. injection of styrene-7,8-oxide, single-strand breaks or alkali-labile sites

in DNA were increased in male NMRI mice (in kidney, liver, lung, testis, and brain)

(Walles and Orsen 1983), male CD-1 mice (in liver, lung, kidney, spleen, and bone

marrow) (Sasaki et al. 1997), female C57BL/6 mice (in liver, lymphocytes, bone marrow,

and kidney) (Vaghef and Hellman 1998), and male ddY mice (in stomach, colon, liver,

kidney, urinary bladder, lung, brain, and bone marrow) (Tsuda et al. 2000). Tsuda et al.

(2000) and Sasaki et al. (1997) took measurements at multiple time points (3 to 24 hours)

and found that DNA damage decreased with time.


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Table 5-8. DNA damage in experimental animals exposed to styrene or styrene-7,8-oxide.

Species (organs) Exposure

(administration) Assay method Result Reference Styrene Male NMRI mice (kidney, liver, lung, testis, and brain)

1.7–10.1 mmol/kg b.w. [177–1,052 mg/kg b.w.] (single i.p. injection)

DNA unwinding; hydroxylapatite separation

+ Walles and Orsen 1983

Female C57BL/6 mice (liver, PBLs, bone marrow, and kidney)

100–500 mg/kg b.w. (single i.p. injection)

comet assay + Vaghef and Hellman 1998

Male NMRI mice (PBLs, bone marrow, and liver)

[175–350 ppm], 6 h/d, 7 d/wk, for 1, 3, 7, or 21 d (inhalation)

comet assay ± Vodicka et al. 2001b

Female Fischer rats (PBLs)

125–500 ppm, 6 h/d, for 2 wk (inhalation)

comet assay – Kligerman et al. 1993

Styrene-7,8-oxide Male NMRI mice (kidney, liver, lung, testis, and brain)

1.8–7 mmol/kg b.w. [216–841 mg/kg] (single i.p. injection)

DNA unwinding; hydroxylapatite separation

+ Walles and Orsen 1983

Male CD-1 mice (liver, lung, kidney, spleen, and bone marrow)

400 mg/kg b.w. (i.p.) comet assay + Sasaki et al. 1997

Female C57BL/6 mice (liver, lymphocytes, bone marrow, and kidney)

50–200 mg/kg b.w. (i.p.) comet assay + Vaghef and Hellman 1998

Male ddY mice (stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow)

400 mg/kg b.w. (i.p.) comet assay + Tsuda et al. 2000

+ = positive; ± = equivocal – = negative.

5.4.3.3 Mutations 1 No studies evaluating specific gene mutations in experimental animals exposed to styrene

or styrene-7,8-oxide were identified.

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7

8

5.4.3.4 Cytogenetic studies 4 Cytogenetic effects include SCE, chromosomal aberrations, micronuclei, aneuploidy, and

polyploidy. In vivo cytogenetic studies of styrene and styrene-7,8-oxide exposure have

been conducted in mice, rats, and hamsters and are reviewed below. Cohen et al. (2002)

reviewed the cytogenetic effects of styrene and styrene-7,8-oxide in experimental animals


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and reported that positive effects were seen only at high exposure levels that are not

likely relevant for human exposure; however, because huyman exposures are usually of

much longer duration, the authors suggested that lower exposure levels over longer

exposure periods could have clastogenic effects in animals.

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Styrene 5 IARC (1994a) and Scott and Preston (1994a) reviewed three to eight studies each for

SCE, chromosomal aberrations, and micronuclei in experimental animals (rats and mice)

exposed to styrene by inhalation, i.p. injection, or gavage. All of the studies gave positive

or weakly positive results for SCE; SCE were detected in liver, alveolar macrophages,

lungs, bone marrow, splenocytes (weakly positive), and lymphocytes of mice and

splenocytes and lymphocytes (weakly positive) of rats. In contrast, all the studies except

one (for each end point) gave negative results for chromosomal aberrations and

micronuclei. Polyploidy was observed in Wistar rat bone-marrow cells following

administration of styrene by inhalation at 300 ppm for 11 weeks. Most studies were of

short duration (≤ 2 weeks). One inhalation study lasted for 12 months but did not report

increased incidences of chromosomal aberrations in rat bone marrow following exposure

to concentrations up to 1,000 ppm. Results from the studies reviewed by IARC (1994a,

2002) and Scott and Preston (1994a) are summarized in Table 5-9.

Only a few studies were identified that examined cytogenetic effects in experimental

animals exposed to styrene and were published after the IARC (1994a) review. IARC

(2002) reviewed one additional study of SCE and chromosomal aberrations in F344 rats

exposed to styrene at 4,260 mg/m3 [1,000 ppm] for 4 weeks. The results were negative.

The genotoxicity of styrene and 1,3-butadiene was evaluated in B6C3F1 mice exposed for

8 hours by inhalation (Leavens et al. 1997). Butadiene-exposed mice exhibited increased

micronuclei in bone marrow, while styrene-exposed mice did not. In another study, male

NMRI mice exposed to styrene at 1,500 mg/m3 [350 ppm] had significantly increased

micronuclei in bone marrow after 7 days of exposure but not after 21 days of exposure

(Vodicka et al. 2001b). However, when this study was repeated, there was no evidence of

clastogenicity (micronuclei or chromosomal aberrations) in male NMRI mice exposed to

styrene at 750 or 1,500 mg/m3 [175 or 350 ppm] for 1, 3, 7, 14, or 21 days [micronuclei


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were evaluated independently by two laboratories] (Engelhardt et al. 2003). The authors

suggested that the positive result in the first experiment might have been the result of

some unidentified experimental variation, because the results were inconsistent between

time points.

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Styrene-7,8-oxide 5 Fewer studies have examined the cytogenetic toxicity of styrene-7,8-oxide (IARC 1994b,

Scott and Preston 1994a). Results are summarized in Table 5-9. IARC (2002) did not

review any additional studies of clastogenic effects in experimental animals exposed to

styrene-7,8-oxide. SCEs were not increased in Chinese hamster bone-marrow cells

following inhalation or i.p. injection of styrene-7,8-oxide or in mouse bone-marrow cells

following inhalation exposure. Positive results for SCE were found in mouse bone-

marrow cells following a single i.p. injection of 100 mg/kg b.w., and weakly positive

results in mouse liver cells and alveolar macrophages following inhalation exposure.

Chromosomal aberrations were increased in mouse bone-marrow cells in two of three

studies, but not in Chinese hamster bone-marrow cells. Only two studies were available

that examined micronuclei in rodents exposed to styrene-7,8-oxide. In both studies, one

in BALB/c mice and the other in Chinese hamsters, micronucleus formation was not

increased in bone-marrow cells following a single i.p. injection of styrene-7,8-oxide at

250 mg/kg b.w.

One of these studies, Sinsheimer et al. (1993), investigated the effects of both the R- and

S-styrene-7,8-oxide isomers when administered by i.p. injection at 100 mg/kg b.w. to

male CD-1 mice. No effect on chromosomal aberrations per cell was reported with either

isomer, but the percentage of cells with SCE increased significantly (P < 0.01) following

exposure to the S-isomer (2.75 ± 0.50, mean ± SD) but not the R-isomer (1.75 ± 0.96),

compared with dimethylsulfoxide (DMSO) solvent controls (1.00 ± 0.82). The number of

SCE per cell was also significantly higher for the S-isomer than for the R-isomer or

DMSO. The mitotic index was significantly lower for both the R-isomer (2.74 ± 0.28)

and the S-isomer (2.58 ± 0.22), compared with controls (3.51 ± 0.30).


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Table 5-9. Cytogenetic effects of styrene and styrene-7,8-oxide in experimental animals

Styrene Styrene-7,8-oxide

End point Species and cell type Results LED/HIDa (mg/kg) Results

LED/HIDa

(mg/kg) mouse bone marrow mouse liver mouse alveolar macrophages mouse lymphocytes mouse lung cells mouse splenocytes

+ + + + +

(+)

500 580 580 450 450 450

± b (+) (+) NT NT NT

100 72 72 NT NT NT

rat splenocytes rat lymphocytes

+ ±

750 225

NT NT

NT NT

SCE

Chinese hamster bone marrow NT NT – 500 mouse bone marrow mouse lung cells mouse lymphocytes mouse splenocytes mouse spermatocytes

– – – –

NT

1,000 900 900 900 NT

+c NT NT NT –

50 NT NT NT 250

rat bone marrow rat lymphocytes

± –

270 450

NT NT

NT NT


Chinese hamster bone marrow – 225 – 500 mouse bone marrow mouse splenocytes mouse erythrocytes

± – –

250 900 900

– NT NT

250 NT NT

rat bone marrow rat lymphocytes

– –

3,000 450

NT NT

NT NT

Micronuclei

Chinese hamster bone marrow – 1,000 – 250 Polyploidy rat bone marrow + 270 NT NT Aneuploidy rat bone marrow – 270 NT NT Sources: IARC 1994a,b, 2002, Scott and Preston 1994a. NT = not tested; + = positive in most studies; (+) = weakly positive; ± = Similar number of positive and negative studies; – = negative in most studies. aLED = lowest effective dose in positive studies; HID = highest ineffective dose in negative studies; LED given for studies with mixed results. b One study was positive for the S-isomer but not the R-isomer. c Positive in 2 of 3 studies.

5.4.4 Studies in styrene-exposed workers 1 Many studies have examined the genetic effects of styrene in human populations;

however, interpretation of these studies is complicated by a number of factors that

increase the likelihood of both false positive and false negative results. These include less

control over study design details than in in vitro studies or animal bioassays, lack of

appropriate exposure data, and the need to control for possible confounding factors, such

as smoking or co-exposure to other chemicals used in the plastics industry (e.g., organic

peroxides, dichloromethane, hydroquinone, dimethylaniline, and maleic anhydride).

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Other important study limitations include relatively small control groups, low sensitivity,

and high interindividual variability. [These facts have an impact on any human

biomonitoring study and, together with interlaboratory differences, may be responsible

for much of the ambiguity and inconsistency apparent in styrene population studies.]

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Exposure to other chemicals that may also cause genetic damage is often correlated with

exposure to styrene. The following factors were considered by Cohen et al. (2002) to

increase the probability that an observed relationship is causal: (1) adequate statistical

control of confounders, (2) a positive dose-response relationship among exposed subjects,

and (3) a positive association across studies between a central measure of exposure and

the average magnitude of the increased frequency of the effect in each study. Results of

studies in styrene-exposed workers are summarized below for DNA adducts, DNA

damage, mutations, and cytogenetic markers.

5.4.4.1 DNA adducts 13 Results from studies in Bohemia (Czech Republic), the United States, and Germany are

summarized in Table 5-10. Very few studies were available on the detection of styrene-

specific DNA adducts in humans before 1994 (IARC 1994a). Liu et al. (1988b) reported

unidentified adducts in 1 styrene-exposed worker, and Vodicka et al. (1993) and Vodicka

and Hemminki (1993) reported O6-guanine adducts in lamination workers.

The two reports of DNA adducts in lamination workers by Vodicka and coworkers were

part of a series of studies (Koskinen et al. 2000a, 1995, Vodicka and Hemminki 1993,

1999, Vodicka et al. 1994) using samples collected from workers at a group of factories

in the same geographic area of Bohemia. [In many cases, the same individuals were

sampled repeatedly, and although the same individuals could be identified across some of

the studies, this was not possible in all cases.] Up to six samples were collected from each

individual between December 1992 and March 1995 (Vodicka et al. 1999). These six

occasions were (I) in December 1992, (II) in July 1993 one day before summer vacation,

(III) in August 1993 on the first day of work after two weeks of vacation, (IV) in

September 1993 after an additional month of work, (V) in February 1994, and (VI) in

March 1995. Data from these samplings are reported in Table 5-10. Two groups of

controls were used in this study: 7 factory controls and 8 laboratory controls (increased to


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13 for sampling VI). The factory controls were sampled on occasions II through V, and

the laboratory controls were sampled on occasions V (N = 8) and VI (N = 13);

sampling VI was reported in the study by Vodicka et al. (1999) and included data for

laboratory controls only. These studies also included measurements of single-strand

breaks in DNA (see Section 5.4.4.2) and HPRT mutations in the same groups of workers

(see Section 5.4.4.3).

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The results for O6-guanine adducts from sampling I were reported in Vodicka et al.

(1993) for 23 hand-lamination workers. The workers were divided into 2 groups that

differed by styrene exposure duration and levels, and adduct levels did not differ between

the controls and either group. The results for samplings II, III, and IV for samples from 7

or 9 workers (see Vodicka et al. 1999) were reported as part of a study of the persistence

of O6-guanine adducts (Vodicka et al. 1994). Vodicka et al. (1995) also reported the

results for O6-guanine adducts from samplings II, III, and IV and added results for

sampling V. [In the Vodicka et al. (1995) publication, the December 1992 results

reported by Vodicka et al. (1993) were not included, and the other samplings were

numbered I through IV.] Levels of styrene-specific DNA adducts were significantly

higher in workers than in controls at all sampling times before and after vacations for

samplings II, III, and IV, but there was no significant difference between samplings for

the exposed workers. Vodicka et al. (1994) therefore concluded that removal of specific

O6-styrene adducts from DNA was very slow. The results of the final sampling (VI) from

this group of workers was reported by Vodicka et al. (1999) together with occupational

exposure data from the earlier samplings. Separate values were reported for 11 workers

and 10 controls (of a total of 13 sampled in each group) and for the 8 workers and an

unspecified number of controls studied in previous samplings.

In the series of six consecutive samplings over 3 years described above, no tendency of

O6-guanine adducts to accumulate was reported, suggesting a well-established

equilibrium between DNA adduct formation and removal in chronically and highly

exposed hand-lamination workers (Vodicka et al. 1999). Although this study did not find

continued accumulation of O6-guanine adducts, Vodicka et al. (1994) interpreted the


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relatively constant levels of these adducts over time, including time away from work for

vacations, as evidence for their persistence.

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In addition to the O6-guanine adducts reported in the studies summarized above, other

types of adducts (e.g., β-N1-adenine, N2-guanine, and 8-hydroxy-2'-deoxyguanosine)

have been measured in styrene-exposed workers (Horvath et al. 1994, Koskinen et al.

2001a, Marczynski et al. 1997a, Rappaport et al. 1996, Vodicka et al. 2003). In studies in

Bohemia, low levels of β-N1-adenine adducts were detected in styrene hand-lamination

workers by a high-performance liquid chromatography–based method, but the adduct

levels were not significantly higher in workers than in controls (Koskinen et al. 2001a,

Vodicka et al. 2003). In studies of U.S. workers, N2-guanine adducts and a second

unidentified adduct were detected in 48 workers of both sexes employed in a boat-

manufacturing facility where mean styrene exposure was 64 mg/m3 [15 ppm] (range = 1

to 235 mg/m3 [0.2 to 55 ppm]) (Horvath et al. 1994, Rappaport et al. 1996). [However,

these studies included no controls.] Marcynski et al. (1997a) reported on 17 styrene-

exposed boat builders in Germany (aged 23 to 60) and 67 age-matched healthy volunteers

without prior exposure to styrene. Levels of 8-hydroxy-2'-deoxyguanosine adducts [an

indicator of oxidatively damaged DNA] were significantly higher in the workers than in

the controls.

Levels of β-N1-adenine adducts were significantly correlated with measures of styrene

exposure, including styrene in exhaled air (r = 0.613, P = 0.007), styrene in blood (r =

0.558, P = 0.003), and urinary mandelic acid (r = 0.836, P = 0.0003) (Koskinen et al.

2001a), and styrene at the workplace (r = 0.730, P < 0.001), styrene in blood (r = 0.605,

P < 0.001), and urinary mandelic acid (r = 0.670, P = 0.001) (Vodicka et al. 2003).

Significant correlations between adducts and styrene exposure were also reported for the

population studied by Horvath et al. (1994) and Rappaport et al. (1996) (for styrene in

workplace air and N2-guanine adducts, r = 0.244, P = 0.049; for unidentified adducts, r =

0.330, P = 0.012).


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Table 5-10. Studies of DNA adducts in white blood cells of workers occupationally exposed to styrene in Bohemia, the United States, and Germany

Exposure indicators (mean)

Adducts/108 nucleotides (mean ± SD)

No. of subjects (exposed/ control)

Mean years

employed (exposed subjects)

Styrene in air [ppm]a

Urinary mandelic acid

(mg/g of creatinine) Adduct type Exposed Controls Reference

Bohemiab Sampling 10/8 13/10

12 6

[86] [50]

380 330

O6-guanine (I) 4.7 ± 1.9c 7.3 ± 4.9c

0.3 ± 0.3 1.1 ± 1.3

Vodicka et al. 1993

9/7 7d/7 9/7

6.7 [28] 157 O6-guanine (II) (III) (IV)

4.9 ± 2.4** 5.1 ± 1.9** 6.0 ± 1.8**

1.4 ± 0.8 0.7 ± 0.3 0.9 ± 0.6

Vodicka et al. 1994

8/7 (factory) 8/8 (laboratory)

9 [21] 146 O6-guanine (V) [4.8 ± 2.5]e*** [0.8 ± 0.4]e [0.2 ± 0.5]e

Vodicka et al. 1995

11/10 8/?

7.2 [16] 187 O6-guanine (VI) 5.9 ± 4.9*** 7.2 ± 4.9***

0.7 ± 0.8 0.8 ± 0.8

Vodicka et al. 1999

9/11 7.8 [18] 106 β-N1-adenine 0.08 ± 0.01 ≤ 0.04f Koskinen et al. 2001a

19/7 14.1 [40] NR β-N1-adenine 0.3 ± 0.05 ≤ 0.04f Vodicka et al. 2003 United States 47/0 at least 1 [15] NR N2-guanine

unidentified 15.8 ± 3.2g

14.2 ± 2.3 no controls no controls

Horvath et al. 1994

Rappaport et al. 1996 Germany 17/67 1– > 10 NR NR 8-OH-2α-

deoxyguanosine 2,230 ± 540*** 1,520 ± 450 Marczynski et al.

1997a **Significantly different from the controls at P < 0.01 by Student’s t-test. ***Significantly different from the controls at P ≤ 0.001 by the Mann-Whitney U test (Vodicka et al. 1995, 1999) or Student’s t-test (Marczynski et al. 1997a). a Values converted from mg/m3 to ppm by multiplying by 0.233 and rounding to 2 significant figures. b The populations overlapped between studies to some extent, as noted in the text; see text for description of samplings.


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c The authors reported that the true differences were larger than calculated because of an unusually high adduct level in one of the controls, no statistical analysis was reported. d Per Vodicka et al. (1995). e Estimated from Figure 2 of Vodicka et al. (1995). f Detection limit; no adducts were detected in controls; no statistical analysis reported. g Mean ± SE.


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5.4.4.2 DNA damage and repair 1 Pero et al. (1982) tested the sensitivity of human lymphocytes to stimulation of UDS by

N-acetoxy-2-acetylaminofluorene (NA-AAF) after exposure to styrene in vivo or in vitro.

UDS resulting from exposure to NA-AAF in vitro was significantly greater in

lymphocytes obtained from workers in a Swedish fiberglass-reinforced polyester plastic

factory exposed to styrene at 1 to 40 ppm than in lymphocytes from workers in a

mechanical industry in the same town. In lymphocyte cultures exposed to styrene at 0 to

750 μM, NA-AAF–induced UDS was increased significantly (P < 0.001) compared with

the mean level of unexposed controls, and there was a significant (P < 0.001) linear

correlation with styrene concentration up to 100 μM, above which the effect remained

elevated. The authors concluded that styrene could make lymphocytes more sensitive to

other genotoxic exposures and suggested that one potential mechanism could be

induction of mixed-function oxygenase activity by styrene, leading to increased

metabolism and activation of genotoxins such as NA-AAF.

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DNA repair capacity was measured in lymphocytes from 14 styrene-exposed boat

builders and 7 controls from the wood manufacturing industry in an X-ray challenge

assay (Oberheitmann et al. 2001). Lymphocytes obtained were exposed to X-rays, and

the rate of exchange-type chromosomal aberrations per 100 metaphases was determined.

However, the duration of radiation exposure was different for the exposed and control

cultures, so the results could not be used for comparison. The authors compared the

results with those for 2 historical control subjects, who were individuals from the

research institute (the authors noted that the comparison could only be exploratory).

Significantly more chromosomal aberrations were found in the lymphocytes from

styrene-exposed workers than in the historical controls. In the exposed group, the

challenge response was significantly correlated with cumulative lifetime exposure to

styrene (years of exposure), but not with the current exposure (measured as styrene in the

blood). The authors concluded that their results were consistent with the hypothesis that

long-term exposure to styrene affects DNA repair activities in humans.

A significant positive correlation was observed between exposure parameters and rates of

base-excision repair (irradiation-specific repair and the repair of oxidatively damaged


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DNA) (Vodicka et al. 2004a). Peripheral lymphocytes from styrene-exposed workers at

three plants or from controls working in a regional hygienic station were compared for

their ability to repair single-strand breaks induced by γ-rays in vitro. Repair rates

(reported as SSB/109 Da) were significantly higher for Plant A (mean ± SD = 0.94 ±

0.32, P = 0.023), Plant B (0.96 ± 0.44, P = 0.016), and Plant C (1.63 ± 0.41, P = 0.001)

than for controls (0.55 ± 0.64). Across the three plants, the rate of DNA repair correlated

significantly (r = 0.308, P = 0.031) with styrene concentration in the blood. DNA repair

increased with increasing styrene air concentration, but differed significantly from the

controls only for the high-exposure group (exposed to styrene at > 50 mg/m3 [12 ppm]; P

= 0.034). The authors suggested that particular DNA repair pathways might be induced

by styrene exposure.

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Slyskova et al. (2007) compared the capacity to repair oxidatively damaged DNA in

mononuclear leukocytes obtained from 24 lamination workers occupationally exposed to

styrene for an average of 14.6 years and 15 unexposed controls. The DNA-repair capacity

was moderately higher in the exposed group compared with the controls, but the

difference was not significant. There was no significant correlation between the DNA-

repair capacity and styrene exposure or biomarkers of genotoxic effects (strand breaks,

DNA adducts, chromosomal aberrations, or HPRT mutant frequencies). The authors

suggested that the lack of a significant difference was most likely related to inter-

individual variability in DNA-repair rates (significant differences were noted for sex and

polymorphisms in GSTM1, XRCC1, and XPC genotypes), differences in the levels and

duration of exposure, and the small sample size.

The results of 13 studies evaluating DNA damage in workers with high levels of styrene

exposure from fiberglass-reinforced-plastics production, boat building, or hand

lamination are summarized in Table 5-11. Twelve studies used peripheral blood

lymphocytes, and one study (Migliore et al. 2002) used sperm cells. All studies included

exposure measures — either styrene in air, mandelic acid (or mandelic acid plus

phenylglyoxylic acid) in urine, or both. In three studies (Godderis et al. 2004, Laffon et

al. 2002a, Maki-Paakkanen et al. 1991), the authors estimated styrene concentrations in

air from urinary mandelic acid levels.


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Higher levels of DNA damage were found in styrene-exposed individuals than in controls

in all of the studies using the DNA unwinding assay (Brenner et al. 1991, Maki-

Paakkanen et al. 1991, Shamy et al. 2002, Walles et al. 1993) and in 6 of the 8 studies

using the comet assay (Buschini et al. 2003, Laffon et al. 2002a, Migliore et al. 2002,

Somorovská et al. 1999, Vodicka et al. 1995, Vodicka et al. 1999). No increase in single-

strand breaks was reported in a study that used nick translation (Holz et al. 1995).

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Some studies found a significant correlation between DNA damage and markers of

styrene exposure (r = 0.753, P < 0.01 for urinary mandelic acid; r = 0.601, P < 0.01 for

urinary phenylglyoxylic acid (Shamy et al. 2002); r = 0.470, P = 0.031 for styrene

concentration at the workplace, and r = 0.545, P = 0.036 for styrene concentration in the

blood (Vodicka et al. 1999); see Section 5.5.3.1 for a description of the studies by

Vodicka et al.). Significant correlations also were found between DNA damage and N-

terminal hemoglobin adducts (partial r = 0.23, P = 0.010) (Godderis et al. 2004) or O6-

guanine DNA adducts (r = 0.719, P = 0.001) (Vodicka et al. 1999). Walles et al. (1993)

reported that single-strand breaks correlated significantly with increasing exposure when

measured at the end of a shift but not at the beginning of a shift. [This study did not use

controls.] Single-strand breaks in sperm cells did not correlate with urinary markers in a

study of hand laminators in Italy, but the urinary samples were taken on a different day

than the semen samples. In contrast to these findings, (Vodicka et al. 2004a) found that

single-strand breaks correlated negatively with most markers of styrene exposure

(r = –0.350, P = 0.007 for styrene in blood; r = –0.402, P = 0.01 for urinary mandelic

acid; r = –0.403, P = 0.001 for urinary phenylglyoxylic acid; and r = –0.375, P = 0.003

for urinary 4-vinylphenol conjugates). As discussed above, Vodicka et al. suggested that

styrene exposure might induce more efficient repair of single-strand breaks because of a

positive correlation between DNA repair capacities and markers of styrene exposure.

All of the studies of DNA damage obtained information on the smoking history of the

subjects, and two studies (Shamy et al. 2002, Vodicka et al. 1995) noted that no smokers

were included in their exposed or control groups. Of the remaining 11 studies, 3 found

that smoking had potentially confounding effects on levels of DNA damage. Brenner et

al. (1991) observed that the number of cigarettes smoked per day significantly increased


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the number of single-strand breaks in the exposed group [no smokers were included in

the control group]. Walles et al. (1993) reported that smoking increased single-strand

breaks in samples taken at the end of a shift, and Laffon et al. (2002a) found an increase

in DNA tail length in the comet assay for smokers in the exposed group. No other

potential confounders were reported to have a significant effect; however, not all studies

included potential confounders in their statistical analyses.

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5

6


Table 5-11. DNA damage (single-strand breaks or alkali-labile sites) in workers occupationally exposed to styrene

Reference Location

Styrene in air (ppm)a Urinary

mandelic acid (mg/g of

creatinine)a

Study population Exposure duration

(mean or range) No. exposed & controls Method and results Comments

Peripheral blood lymphocytes Brenner et al. 1991

U.S.

air: 0.6–44 urine: 244

fiberglass-reinforced boat building 2.7 yr 14 exposed 9 controls

DNA unwinding, hydroxylapatite sep. negative log of fraction of double-stranded DNA exposed 0.025 ± 0.02** control 0.15 ± 0.01

More smokers (43%) among workers than controls (0%), but reverse for ex-smokers (21% exposed; 55% controls) Workers also exposed to acetone [nongenotoxic] and methylene chloride

Maki-Paakkanen et al. 1991

NR

air: [70]b

urine: 9.4 mmol/L reinforced-plastics production 6.7 yr 9 exposed 8 controls

DNA unwinding, hydroxylapatite sep. negative log of fraction of double-stranded DNA exposed 0.13 ± 0.04* control 0.09 ± 0.02

Authors reported that other variables were considered to exclude their effects on the results: age, sex, health status, recent viral infections, vaccinations, exposure to possible mutagenic chemicals, alcohol consumption, and drug intake; however, statistical analyses do not appear to include these variables

Walles et al. 1993

Sweden

air: 0.4–20 urine: ND–261

plastics factory 0–25 yr 17 exposed 0 controls

Alkaline elution normalized area above elution curve Time relative to shift before 33 x 10-3/h* end 41 x 10-3/h correlated significantly with increasing exposure at end of shift but not before

Highest levels seen in one man who had taken paracetamol, which has increased single-strand breaks in mice

9/29/08 299


300 9/29/08

Reference Location



creatinine)a



Holz et al. 1995

Former German Democratic Republic

air: [0.017–0.82] urine: 43.9 ± 31.5

styrene production plant 18 yr 25 exposed 25 controls

Nick translation cpm of radioactivity incorporated exposed 2,370 ± 1,358 control 2,550 ± 988

Significantly (P < 0.01) higher styrene, ethylbenzene, benzene, and toluene in exhaled air of exposed workers than controls Subjects and controls matched for age and sex; similar smoking habits confirmed by plasma cotinine; higher self-reported alcohol consumption in controls

Vodicka et al. 1995

Bohemia

air: [21–28] urine: 146 ± 77

hand laminators 9 yr 9 exposed 15 controls

Comet: tail moment Abnormal cells exposed 5.50 ± 3.04* control 1.00 ± 3.41 tail length and % DNA in tail also significant Total cells: NS

All subjects were nonsmokers

Vodicka et al. 1999

Bohemia

air: [16–38] urine: 161–351

hand laminators 7.2 yr 13 exposed 13 controls

Comet: tail moment exposed 1.9 ± 0.8*** control 0.6 ± 0.2 tail length and % DNA in tail also significant except for exposed vs. control smokers

Difference between exposed and controls also significant when smokers (P < 0.019; 4 exposed/5 control) and nonsmokers (P < 0.005; 9 exposed/8 control) were considered separately

Somorovská et al. 1999

Bohemia

air: high: [46 ± 24] med: [13 ± 5.3] urine: NR

hand laminators and sprayersc 14.0 yr 17 high exposure (hand laminators) 12 med. exposure (sprayers)19 controls

Comet: % DNA in taild high-exposure [30 ± 9]*** medium-exposure [27 ± 8]*** control [14 ± 5]

Styrene levels almost 4 times as high in the high-exposure than medium-exposure group, but no difference in SSBs


9/29/08 301

Reference Location



creatinine)a



Laffon et al. 2002a

Spain

air: 17–19b

urine: 313–353 fiberglass-reinforced-plastics factory 17 yr 14 exposed 30 controls

Comet: tail length exposed 48.68 ± 0.33** control 43.34 ± 0.18

Smoking significantly increased tail length in exposed but not controls; smoking time related to age and styrene exposure duration Influence of exposure duration, age, smoking, and GSTM1 and GSTT1 genotype included in analysis of variance; other possible confounders considered in interviews (alcohol consumption, medication, medical diagnostic tests, previous occupational exposure to chemicals); however, they do not appear to have been included in the statistical analysis Exposure to other possible genotoxins [organic peroxides, acetone, and dichloromethane] possible, but not evaluated

Shamy et al. 2002

Egypt

air: NR urine: 90–170

reinforced plastic plant 20 yr 26 exposed 26 controls

DNA unwinding assay hydroxyapatite separation % DNA with SSBs, median (range) exposed 40 (22–65)** control 10 (6.5–13) Exposure-response with urinary markers (r) mandelic acid 0.754*** phenylglyoxylic acid 0.601***


302 9/29/08

Reference Location



creatinine)a



Buschini et al. 2003

Italy

air: 36.8 ± 0.7 urine: 206 ± 2.4e

polyester resin production; glass-fiber-reinforced plastic manufacture 8.5 yr 48 exposed 14 controls

Comet: tail moment 99th percentile exposed 34.1 ± 14.0*** control 12.4 ± 4.9 median: NS

Controls were of comparable age and sex


Bohemia

air: [19 ± 13.1] urine: 206 ± 2.4e

fiberglass-reinforced plastic manufacture 4 yr 86 exposed 16 plant controls 26 external controls

Comet: SSBs per 109 Da exposed 0.29 ± 0.21 plant control 0.57 ± 0.26 external control 0.53 ± 0.26 correlation (r), P-value Blood: styrene: –0.350, 0.007 Urinary: mandelic acid: –0.402, 0.01 phenylglyoxylic acid –0.403, 0.001 4-vinylphenol conj. –0.375, 0.003

Exposed subjects almost 9 years younger than controls and included more men (71% vs. 52%) and more smokers (51% vs. 19%)

Godderis et al. 2004

Belgium

air: 9.5 ± 9.6b

urine: 202 ± 148 laminators 14.2 yr 44 exposed 44 controls

Comet: % DNA in tail exposed 0.80 ± 0.31 control 0.80 ± 0.34

Controls matched for age and smoking habits and recruited from 2 plants manufacturing electrical wires and telecommunications cables


9/29/08 303

Reference Location



creatinine)a



Sperm cells Migliore et al. 2002

Italy

air: NR urine: 202 ± 148

hand laminators 9.2 years 46 exposed 27 controls

Comet: tail moment exposed 1.5 ± 0.6*** control 0.8 ± 0.4 % DNA in tail: significant*** Exposure-response with urinary markers: NS

More smokers (63.0%) in control group than in exposed (48.2%) Semen samples (for SSB analysis) and urine samples taken on different days

C = controls, E = exposed, ext. = external; HA = hydroxylapatite; MA = urinary mandelic acid; med= medium, ND = not detected; NR = not reported, PGA = urinary phenylglyoxylic acid; 4-VPT = urinary 4-vinylphenol conjugates. a Mean ± SD or range; air concentrations in brackets converted from mg/m3 to ppm (1 mg/m3 ≈ 0.23 ppm). b Calculated from urine mandelic acid levels by the study authors. c Study population also included workers with low exposure (maintenance workers), but these were not included in the analysis. d Values estimated from graph. e Mandelic acid + phenylglyoxylic acid.


304 9/29/08

5.4.4.3 Mutations 1 Studies evaluating mutation frequency (for HPRT or glycophorin A [GPA] genes) in

styrene-exposed workers are summarized in Table 5-12. Mutations at the HPRT locus

may be associated with a number of other factors (e.g., different types of T cells with

different lifespans, host polymorphisms affecting metabolism and DNA repair, and

background exposures, such as food intake or smoking) (Vodicka et al. 1995).

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Tates et al. (1994) reported that HPRT mutation frequency was higher among 46 workers

from the former German Democratic Republic who had been exposed to styrene and

dichloromethane than in 5 controls [mutation frequency could be measured for only 5 of

23 controls]. Controls were matched by age, sex, and smoking status [No other

information was provided on the control subjects.].

A series of studies measuring HPRT mutation frequency in a relatively small number of

lamination workers from Bohemian hand-lamination factories (with up to six samplings

for each individual) was conducted during a three-year period by Vodicka and co-

workers and reported in two publications (Vodicka et al. 1995, 1999) (see also the

description of this population in Section 5.4.4.1), and another study of workers with

differing levels of exposure to styrene in these factories was published by Somorovská et

al. (1999) and Vodicka et al. (2001a). Results for HPRT mutation frequency from all of

these studies were also summarized and reanalyzed in two reviews of styrene

genotoxicity by Vodicka et al. (2002a, 2003). The 2003 analysis of all of the samplings

from this population (Vodicka et al. 1995, 2001a, 1999) found a higher HPRT mutation

frequency in styrene-exposed workers (19.8 ± 20.1 per 106 cells) than in controls (14.9 ±

7.7), but the difference was not significant (P = 0.656). Some of the individual samplings

did result in significant differences between exposed workers and controls; HPRT

mutation frequency was significantly higher in workers than external controls but not

factory controls in one of four samplings in the 1995 study and in the sixth sampling in

the 1999 study (P = 0.039) (Vodicka et al. 1999). In the 2001 study of these workers,

HPRT mutations were higher in styrene-exposed workers than controls, though not

significantly so. Although Vodicka et al. (2003) reported that their analysis of all data

from the hand-lamination workers did not show a significant difference between exposed

RoC Background Document for Syrene

9/29/08 305

workers and controls, they did find a significant correlation (r = 0.588, P = 0.001)

between HPRT mutation frequency and cumulative exposure [the product of an arbitrary

exposure level and years of employment]. Individual studies had also shown significant

correlations between HPRT mutation frequency and styrene concentration in air, styrene

concentration in blood, urinary mandelic acid, hemoglobin adducts, years of employment,

age of employees, or heterozygosity in the CYP2E1 and GSTP1 genes (Vodicka et al.

2001a, Vodicka et al. 1999). None of the studies reviewed reported a significant

correlation between DNA adducts and HPRT mutation frequency.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Somatic mutations at the GPA locus in erythrocytes were measured in 47 workers from

10 reinforced-plastics plants in Finland (Bigbee et al. 1996). The controls were 47

unexposed individuals matched for age, gender, and smoking status. All exposed and

control subjects had the GPA M/N heterozygous genotype. GPA variant frequencies

reflecting allele loss (φ/N) or allele loss and duplication of the remaining allele (N/N)

were examined. Styrene exposure did not affect φ/N frequency, but N/N frequency was

higher among workers than controls (P = 0.058). When workers were classified into low-

and high-exposure groups, the N/N frequency was significantly higher (P = 0.036) in the

high-exposure group, based on a multivariate analysis of covariance model. However, the

significant difference was seen only when one individual in the high-exposure group with

an exceptionally low value was excluded; when that individual was included in the

analysis, no difference was found. Significant increases in both φ/N and N/N frequencies

also were seen among active smokers, but the analysis for styrene exposure was adjusted

for smoking. The authors concluded that occupational exposure to styrene in the

reinforced-plastics industry resulted in mutagenic effects.


Table 5-12. Mutation frequencies in workers exposed to styrene Reference, industry, & mutation Group (no. M/F)

Years exposeda


Mutation frequency per 106 cells ± SD Comments

Germany Tates et al. 1994 Production of containers & boards with polyester resin (19% –33% styrene) HPRT

5 control (NR) exposed: 24 group I (16/8) 22 group II (9/13) 46 total (25/21)

0 19 (4–31) 20 (8–31) 20 (4–31)

0 [20 (0–140)] [12 (0–34)] [16 (0–140)]

8.6 ± 1.2

15.9 ± 21.1 12.7 ± 6.8 14.3 ± 15.7

Only 5 of 23 control samples could be analyzed because of losses during transport Groups I & II sampled 1 wk apart Workers also exposed to dichloromethane

Bohemia (some overlap in subjects among studies; hand laminators from same plants)

7 factory control (3/4) 9 exposed (2/7)

NR

9 (1.5–17) 0 [21 (5.8–58)]b

15.7 ± 8.3 17.5 ± 12.3 4 samplings combined

Vodicka et al. 1995 Hand-lamination plant HPRT

8 external control (0/8) 9 exposed (2/7)

0

9 (1.5–17) 0 [21 (5.8–58)]b

11.8 ± 6.8 18.0 ± 5.2* sampling IVc

Factory controls (but not external controls) had measurable levels of styrene-specific DNA adducts, suggesting possible low-level exposure Significant difference seen in only 1 of 4 samplings of the same individuals

Vodicka et al. 1999 Hand-lamination plant HPRT

13 external control (3/10) 12 exposed (4/8)

0 7.2 (2–17)

0 [16 (3.5–36)]

14.2 ± 6.5 22.3 ± 10.6* sampling VI

Only external controls used

Somorovská et al. 1999, Vodicka et al. 2001a Plastics lamination plant (hand-lamination workers) HPRT

19 control (8/11) 19 exposed (2/17)

0 14 ± 6.1

0 [23 ± 23.5]d

13.3 ± 6.3 20.2 ± 25.8

Controls were clerks in the same factory Mutation frequency significantly higher (P = 0.04) in smokers than nonsmokers overall but not among controls or exposed analyzed separately

306 9/29/08


9/29/08 307

Reference, industry, & mutation Group (no. M/F)

Years exposeda


Mutation frequency per 106 cells ± SD Comments

Finland

Bigbee et al. 1996 Reinforced-plastics workers GPA φ/N

47 control (23/24) 47 all exposed (23/24) 28 highest exp. (NR) 19 lowest exp. (NR)

0 8.5 ± 6.6 NR NR

0 [36 ± 27] [≥ 20] [0.2–19]

8.1e 7.2e 7.6e 7.5e

Multivariate analysis of data adjusted for age, gender, smoking status, and styrene exposure for both φ/N and N/N

GPA N/N 47 control (23/24) 47 all exposed (23/24) 28 highest exp. (NR) 19 lowest exp. (NR)

0 8.5 ± 6.6 NR NR

0 [36 ± 27] [≥ 20] [0.2–19]

5.0e 6.3e 7.2e* 6.0e

Difference nonsignificant for high-exposure group when 1 subject with an exceptionally low value was included

F = female; M = male; NR = not reported. *Significantly different from the control group at P < 0.05 by the Mann-Whitney U test (Vodicka et al. 1995, 1999) or multivariate analysis of variance (Bigbee et al. 1996). a [Mean and range; air concentrations in brackets converted from mg/m3 to ppm (1 mg/m3 ≈ 0.23 ppm).] b Air concentrations measured on the day of sampling. c Listed as sampling IV (Vodicka et al. 1995), which corresponds to sampling V in Vodicka et al. (1999). d Based on mean for all exposed workers; data were not provided for the subset of workers used to study HPRT mutations. e Least squares mean of log-transformed data adjusted for age, gender, and smoking status.


308 9/29/08

5.4.4.4 Cytogenetic markers 1 Cytogenetic markers include chromosomal aberrations, micronuclei, and SCE. The

cytogenetic effects of occupational exposure to styrene have been reviewed (Bonassi et

al. 1996, Cohen et al. 2002, Henderson and Speit 2005, Vodicka et al. 2006b).

Guidelines for monitoring of genotoxic effects in humans are available in Albertini et al.

(2000). Many of the studies reviewed in this section evaluated more than one cytogenetic

marker; however, results are discussed separately for each marker.

2

3

4

5

6

7

8

9

10

11

13

14

15

16

Most of the reviewed studies used questionnaires to gather information on exposed and

referent population characteristics such as age, sex, socio-economic status, disease status,

smoking habits, vaccinations, and past or current exposures to other clastogenic agents,

including X-rays.

Chromosomal aberrations 12 Structural chromosomal aberrations were measured in lymphocytes from styrene-exposed

workers in 31 studies. Details on the study population, exposure levels, study design, and

results for structural chromosomal aberrations are summarized in Table 5-13 and the

findings are summarized after the tables.

.


Table 5-13. Chromosomal aberrations in lymphocytes from workers occupationally exposed to styrene Styrene exposure

Mean (range) Reference (location)

Study populationa

(yrs employed) Number of subjects Air

(ppm)b Urinary MA

(mg/g creatin.)

Results (% cells with CA)c

Exposure response Commentsd Meretoja et al. 1977 (Finland)

Polyester plastic manufacturing workers ― 3 plants (laminators) (0.6–8.5 yr)

Exposed 10 Controls 5

NR

[721 (23–3,257)]

Gapse [0.3] [0.2]

Breakse [16.3]*** [1.6]

Unmatched controls but similar age range; all subjects were male No previous exposure to known clastogenic agents; no recent viral infections or vaccinations

Cell harvest at 64–68 h No dose-response analysis Statistics: Student’s t-test

Meretoja et al. 1978a (Finland)

Polyester plastic manufacturing workers ― 2 plants (laminators) (1−15 yr)

Exposed 1976 16 1977 10

Controls 6

NR (≤ 300) NR (≤ 300)

[570 (23–3,257)] [329 (52–1,646)]

Total 15.1*** 16.2***

2.0

Unmatched controls but similar age range; all subjects were male 10 exposed subjects first samples in 1976 were reanalyzed in 1977 No previous exposure to clastogenic agents No correlation with smoking.

Cell harvest at 64–68 h CAs without gaps not reported. Chromosome-type breaks most common among exposed while aneuploidy was most common in controls Statistics: Student’s t-test

Fleig and Thiess 1978, Thiess and Fleig 1978 (Germany)

Group A: styrene plant (14−25 yr); Group B: poly-styrene plant (3−39 yr); Group 3: three unsaturated polyester resin plants (2−24 yr)

Exposed group A 5 group B 12 group C 14

Controls 20

NR NR (0.01–0.53)f NR (50–300)g

(mg/L) NR (19–40) NR (< 5–100) NR (102–> 1,500)

Total 3.8 5.1 9.2*

5.5

w/o Gaps 1.6 1.9 5.3*

2.1

Group B and controls were male, gender not identified for other groups. Mean age similar for all groups Workers also exposed to peroxides, styrene-7,8-oxide, methylene chloride, and acetone

Cell harvest at 70–72 h CA reported as including and excluding gaps but types of CAs in these categories were not defined; polyploid cells counted separately

9/29/08 309


310 9/29/08

Styrene exposure Mean (range)

Reference (location)

Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Statistics: Method and P values not identified

(CA/100 cells) Total 10.2*** 4.9

Breaks 6.9** 2.5

Hogstedt et al. 1979 (Sweden)

Fiberglass-reinforced polyester resin boat manufacture workers (0.5−10 yr)


NR [11.5–92]

490 (225–2,100)

Gaps alone and the sum of gaps, breaks and hyperdiploidy were also significantly higher in exposed than controls

Controls from nearby paper factory matched on age and smoking; all subjects were male Workers exposed to phthalic acid and maleic acid anhydride, propylene glycol, methylethyl ketone peroxide, acetone and cobalt salt Cell harvest at 72 h Total in this table includes breaks and gaps; hyperdiploidy was also scored. Total aberrations not related to exposure time Statistics: Mann-Whitney U-test, one-tailed

(CA/100 cells) Gapsh 6.1 6.6 5.8

3.8

w/o Gaps 7.9*** 7.8*** 8.0***

3.2

Andersson et al. 1980 (Sweden)

Reinforced plastic boat factory workers (0.3−12 yr)

Exposed total 36 high 14 low 22

Controls 37

(mg/m3 × yr) 575 (6–1589) 1204 (710–1589) 137 (6–283)

NR

Increase in frequency of all types of CA measured in exposed compared with controls.

Exposure response Cumulative styrene exposure (time vs. air levels) Low exposure group: (r = 0.58): significant High-dose group: not significant

Total population included 39 exposed and 41 controls but results not available for all subjects Age-matched controls included office, assembly shop, and workshop employees; all subjects were male Also exposed to methylethyl ketone peroxide Cell harvest at 68 h Chromosomal aberrations included gaps (not included in total), breaks, minutes, dicentrics, rings, and acentric fragments; chromatid breaks were most frequent Negative correlation with smoking in controls Statistics: t-test, multiple regression including CA frequency, employment duration, styrene exposure, smoking, alcohol intake, exposure to X-rays and solvents, and use of a breathing mask


9/29/08 311



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Thiess et al. 1980 (Germany)

Polyester resin processing workers (4–27 yr)

Exposed 24 laboratory pilot plant

Controls 24

6 (1−11.5) 58 (0.7−178)

(mg/L) 0–320

Gaps 5.1 NR NR

3.8

w/o Gaps 1.9 NR NR

1.5

Controls: Occupational Health and Protection Department, office staff and plant maintenance workers. Gender not identified Smoking and alcohol habits, virus disease and consumption of drugs were recorded.

Cell harvest at 70–72 h Authors defined “w/o gaps” to include breaks, fragments, chromatid interchanges and dicentric chromosomes; “including gaps” to include both chromatid and isochromatid gaps, but it was not clear if this group also included other CA because it was called “including gaps” by study authors Statistics: Fisher-Yates exact test

Watanabe et al. 1981 (Japan)

Group 1: Reinforced-plastics boat workers Group 2: Polyester resin board workers (0.6−9.3 yr)

Group 1 exposed 9 controls 5

Group 2 exposed 7 controls 8

< 70 (1–211)

36 (NR)

(mg/L) 647 (90–4,300) 32 (5–115)

526 (300–1,360) 32 (5–115)

Total 3.3 3.6

3.6 2.9

Controls matched on age and sex; Group 1 subjects were male; Group 2 subjects included both male and female Exposure varied depending on the work in workshop 1 but was stable in workshop 2 CA scored in M2 cells (< 50/person), most aberrations were gaps. Mitomycin C treatment did not increase the number of CA in exposed or controls Statistics: t-test or Chi-square

(Watanabe et al. 1983) (Japan)

Male fiber-reinforced-plastics boat factory workers in 2 workshops

Exposed total 18 group A 10 group B 8

Controls 6

40–50 (NR)

(mg/L) 332 (0–1,041) 399 (0–1,041) 249 (8–999)

Total 6.5i 6.6 6.4

4.7

w/o Gaps 1.1 1.0 1.3

1.1

All subjects were male, controls matched according to age Smokers: 72% exposed and 50% controls Most aberrations were gaps but also included


312 9/29/08



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd (1 mo–30 yr) Exposure response

Styrene air levels, urinary levels and exposure duration: no relationship

acentric fragments, deletions, and breaks Cell harvest at 50 h No difference in CA between smokers and non-smokers in exposed or control groups Statistics: Mann-Whitney U test and t-test (2-tailed)

Gaps 26.9* 14.4

Breaks 6.8 0

Dolmierski et al. 1983 (NR)

Laminators (1–30 yr)


NR [< 23]

NR

Exposure response Length of employment: no correlation

Little information on exposed or control subjects. Gender was not identified, only 2 controls (ages 22 and 28); ages in exposed ranged from 22–58 yr Exposure was “haphazard” and measured once a year; repeated on 6 subjects after 1.5 yr Cell harvest at 68 h ~40 metaphases/person examined Gaps most common but were not measured in a group of 6 subjects because “interpretation” was difficult Statistics: Poisson’s distribution


9/29/08 313



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Total 30** 9

23*** 8

24*** 8.4

26*** 8.4

32*** 7

39** 6.5

37** 8

25*** 4

44*** 4.5

Camurri et al. 1983, Camurri et al. 1984 (Italy)

Reinforced unsaturated polyester resin manufacturing workers in 9 plants (1–22 yr)

Plant 1 3 Controls 3









NR [7–9]

NR [16–23]

NR [23–34.5]

NR [34.5–46]

NR [46–57.5]

NR [57.5–69]

NR [69–80.5]

NR [80.5–92]

NR (> 92)

(mg/L) NR (45–75)

NR (65–133)

NR (170–694)

NR (151–786)

NR (340–671)

NR (615–777)

NR (489–828)

NR (504–909)

NR (389–1,108)

Exposure response (all subjects) Styrene air concentrations and urinary metabolites: Linear increase (P < 0.01) in 1983 study but not in 1984 study

Data described for 6 plants in 1983 publication, all data described in 1984 publication Controls matched for age, sex, and smoking. No subjects had recent viral infections, vaccinations or exposure to known clastogenic agents; however, processing of unsaturated polyester resins in the reinforced-plastics industry involves exposure to other industrial chemicals (e.g., organic peroxides, solvents, and dyes) Cell harvest at 50 h CA did not correlate with smoking habits

32–360 metaphases/subject (1983 publication) Types of CAs were not reported and it is not clear whether gaps were included in total Statistics: Student’s t-test, linear regression

Hansteen et al. Glass-fiber (CA/100 cells) Controls matched for sex, age and smoking;


314 9/29/08



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd 1984 (Norway)

reinforced polyester plant workers (NR)

Exposed Total 18 group 1 11 group 2 7

Controls 9

13.2 (2–44) 7.5 (2–13) 22.3 (14–44)

NR (200–1,200)j

Gaps 19.5*** 17.6 22.4***

13.4

w/o Gaps 1.2 1.1 1.4

1.7

exposed divided into 2 groups based on exposure

Cell harvest at 48–53 h CA breaks were significantly higher in smokers vs. non-smokers. The group w/o gaps was labeled as “breaks” by the authors and included breaks, fragments and exchanges No differences between 2 exposed groups Statistics: Significant by Fisher-Irwin test but not by Wilcoxon’s 2 sample ranking test; P values reported for total group for gaps only

(CA/100 cells) Nordenson and Beckman 1984 (Sweden)

Glass-fiber reinforced polyester plant workers (1–26 yr)

Total exposed 15 controls 13

Smokers exposed 4 controls 3

Nonsmokers exposed 11 controls 10

24 (NR)

(mM) NR (< 2)

Total 2.8 2.7

4.6 2.7

2.1 2.7

Breaksk [0.4] [0.4]

[0.6] [0.3]

[0.3] [0.4]

All subjects were male; controls were salesmen or office workers with similar smoking habits, and similar age distribution. Exposed to acetone

Cell harvest at 64–68 h Total includes gaps, chromatid breaks and chromosome breaks. Most aberrations were gaps. No correlation with exposure time (type of analysis not reported). Statistical methods: Fisher’s exact test

van Sittert and Propylene (CA/100 cells) All subjects were male


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Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Gaps 1.33 4.36* 0.84

1.32 0.78

Breaks 0.65 1.47* 0.60

0.33 0.44

de Jong 1985 (Netherlands)

oxide and styrene manufacturing workers

No significant difference in exchange-type CA was found between exposed and controls

and 20 subjects not involved in manufacturing (matched by age and smoking status). Samples taken in workers 1 yr (1979), 2 yr (1980), and 3 yr (1981) after exposure; fewer exposed workers each year due to transfer to other plants Workers exposed to propylene oxide and benzene Authors reported no change in styrene, propylene oxide, and benzene air levels from 1978–1981, thus they did not think the increase in CA in 1980 was due to occupational exposure Statistics: methods not reported

Pohlova and Sram 1985 (Czech Republic)

Plant A: polystyrene vessels Plant B: sports boats, plastics (1–11 yr)

Plant A June 36 November 34 Controls 19

Plant B June 22 Controls 22

January 19 Controls 17

[16–35 (1.4–226)]

[NR (9–126)]

(μg/L) NR (35–510) NR (88–972)

NR (40–1140)

NR (200–3,000)

% AB.C 1.38 1.41 1.26

1.72 1.36

2.81 1.88

(CA/100) Gaps 1.36 2.56 1.63

3.23* 1.78

2.59 2.29

Controls matched for sex and age. Smoking and drug intake were similar for all groups. Subjects not exposed to other known mutagens (queried about viral infections, drug intake, X-rays, smoking and alcohol use) CA measured twice: June and November (Plant A) and June and January (Plant B); there was no concurrent control for November sampling in Plant A Cell harvest at 54 h Percent aberrant cells (% AB.C) included cells with breaks and exchanges. Results for gaps only provided per 100 cells. Inter-sampling differences: Plant A – significant increase (P < 0.01) in rates of gaps (from 1st sampling to 2nd sampling) in styrene-exposed workers. Plant B – significantly higher rate of


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Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd gaps at first sampling and significantly higher rate of % AB.C at 2nd sampling in exposed workers No significant differences found for drug intake, gender, and smoking Statistics: Student’s t-test

Maki-Paakkanen 1987 (Finland)

Reinforced-plastics workers (mainly laminators) (1–25 yr)


[23 (8–60)]

(mM) 2.0 (0–7.3)

Total 4.5 4.9

w/o Gaps 3.0 3.7

Controls matched according to sex and smoking. Control and exposed subjects were similar in alcohol and drug intake, vaccinations, recent viral infections, and previous occupational exposure to chemicals Exposed group was mainly laminators Cell harvest at 50 h CA included gaps (most frequent), breaks (mainly chromatid type), and rearrangements (infrequent). CA slightly higher (not significant) in smokers than non-smokers among controls. No correlation with exposure extent or duration Statistics: Student’s t-test

Forni et al. 1988 (Italy)

(A) Reinforced-plastics workers (18.8 yr) (B) Plastic boat manufacturing workers (4.5 yr)

Factory A exposed 32 controls 32

Factory B exposed 8 controls 8

NR [0.4–57]

NR [9.4–45.5]

NR

Total 3.2 2.9

4.3 2.9

w/o Gaps 2.3* 1.6

2.5 1.5

All factory workers were male Controls lived in the same industrial area (gender not identified) matched for age and smoking Subjects not exposed to other known genotoxic agents (radiation, chemicals, drug intake, and


9/29/08 317



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Factory A workers had significantly higher frequency of unstable CA

recent viral infections); workers exposed to low levels of acetone. Cell harvest at 48 h CAs included gaps, breaks, exchanges and unstable chromosome-type aberrations (acentric fragments, dicentrics, and ring chromosomes) Statistics: Wilcoxon matched-pairs test

Jablonicka et al. 1988 (Czech Republic)

Laminated plastic shop workers (11 yr)


[58 (27–134)]

(μL/mM)m NR (214–711)

% AB.C 1.3 1.4

(CA/100) Gaps [0.27]n [0.55]

All subjects were female Controls and exposed subjects had similar age, social habits, living and working environments. Histories of viral infections and other diseases during past 3 mo along with drug and alcohol intake, smoking, and X-ray exposure recorded Subjects with illness or taking medications were excluded; only 2 weak smokers in each group % AB.C included chromosome and chromatid breaks and chromatid exchanges (no exchanges observed), the total number of gaps observed in 1,100 metaphases were reported. Gaps were not considered as aberrations

Cell harvest at 50–52 h (approximately 82% first-division cells) Statistics: Student’s t-test

(CA/100 cells) (Hagmar et al. 1989) (Sweden)

Glass reinforced polyester plastic workers


[13 (0.9–127)]

NR

w/o Gaps 1.2 1.5

Gaps 0.7* 1.7

Total population included 20 workers and 22 controls; technical difficulty prevented analysis on all subjects


318 9/29/08



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd (0.1–25.4 yr)

Exposure response No association with years of employment

One exposed subject was female More smokers in controls (64%) than exposed workers (45%). Similar number of exposed and control subjects reported recent exposure to X-rays (65% vs. 55%) and regular prescription drug use (15% vs. 23%) Workers exposed to low levels of acetone and methylene chloride Cell harvest at 48 h The group “w/o gaps” was labeled as “breaks” by the authors, but included chromatid and isochromatid breaks, pericentric inversions, rings, and dicentrics. Statistics: data were adjusted for age and smoking and transformed using the average square root. Multiple linear regression evaluated employment time, smoking, and age.

Total 5.3 5.7

4.9 6.8

6.0* 3.7

w/o Gaps 3.0 3.1

3.0 3.4

3.0 2.7

Maki-Paakkanen et al. 1991 (Finland)

Reinforced plastic workers (controls from research institute) (smokers – 6.4 yr, non-smokers – 7.2 yr)




[~ 70] (NR)o

(mM) 9.4 (< 1–21.5)

11.0 (< 1–16.6)

6.5 (< 1–21.5)

Exposure response Exposure duration: r = 0.59, P < 0.02

Controls selected from a research institute Age, sex, smoking, health status, alcohol and drug intake, viral infections, vaccinations, and exposure to other chemicals were considered. CA significantly increased in control smokers compared with control non-smokers CA types not defined except to distinguish CA with gaps and CA without gaps Cell harvest at 50 h Statistics: Wilcoxon rank-sum test (one-tailed testing) and t-test (one-tailed)


9/29/08 319



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd w/o Gaps 1.8 1.9

2.4 1.4

1.8 1.6

NR NR

Sorsa et al. 1991 (Finland)

Reinforced plastic industry workers from 32 enterprises (NR)

Past exp. (index pt)Laminators low 22 high 28

Other workers low 11 high 14

Controls plastics plant 12 other factory 42

Pop with CA data Exposed 109 Controls 54

43 (5–182)

11 (1–133)

(mM) 2.2 (NR)j,p

Exposure response: Current or past exposure: no correlation

Total population consisted of 248 exposed workers, including 154 laminators and 63 controls. CA results available for a subset. Inadequate description of the study population. Exposure groups were divided into two subsets based on estimated past exposure index points (calculated from exposure duration, urinary metabolites, and styrene concentrations). Past exposure index not available on all subjects Cell harvest at 50 h Types of CAs not reported except to state that they did not include gaps Age (P = 0.06) and smoking (P = 0.08) were correlated with CA Statistics: regression analysis, no details provided

w/o Gaps 1.4 1.4

3.0* 0.8

Tomanin et al. 1992 (Italy)

Polyester resin workers at 2 factories producing fiberglass tanks (1–18 yr) or fiberglass boats (1.5–15 yr)

Factory 1 (low) exposed 7 controls 7

Factory 2 (high) exposed 11 controls 11

NR [4.8–23]

NR [26–100]

186 (46–345)

725 (423–1,325)

Exposure response Exposure duration: no correlation

Controls matched for sex, age, and smoking Cell harvest at 48 h CA including breaks, dicentrics and other exchanges; gaps not reported No significant effects of smoking Statistics: Mann-Whitney U test (2-sided), and linear regression


320 9/29/08



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Total 2.9 *** 3.0*** 2.8***

0.9

(CA/100) w/o Gaps 2.0*** 2.1*** 1.9***

0.4

Tates et al. 1994 (Germany)

Polyester resin/fiberglass plastic products production workers (4–31 yr)

Exposed total 46 group 1 24 group 2 22

Controls 23

[16 (0–138)] [20 (0–138)] [12 (0–34)]

NR

Exposure response Styrene air levels (TWA: Positive correlation with styrene exposure in Group 1 workers only

Controls matched for age, sex and smoking Workers divided into 2 groups with similar working conditions but blood samples collected 1 wk apart Culture time not stated CA consisted of gaps, iso-gaps, chromatid breaks, isochromatid breaks and fragments; chromatid exchanges were rare. Workers also exposed to dichloromethane (a genotoxin), which was associated with CA in group 1 and the total population No significant differences for chromosomal aberrations between smokers and nonsmokers. Statistics: one-tailed Mann-Whitney U test and bivariate regression analysis

(CA/100 cells) w/o Gaps 2.8 4.0**

2.1

Artuso et al. 1995 (Italy)

Fiber-reinforced plastic boat building workers (NR)

Exposed low 23 high 23

Controls 51

NR [0.5–28] NR [20–319]

NR

Regression analysis (RR, 95% CI) Low: 1.38 (0.98–1.94) High: 1.71 (1.25–2.33)

All subjects were male; controls lived in same geographic area and had similar ages and smoking habits as exposed Carpenters in the low-exposure group also exposed to solvents and wood dust CA analyzed by 2 different labs and 3 slide readers Cell harvest at 72 h CAs included breaks and exchanges; gaps scored but not reported Smoking, alcohol, and exposure to X-rays were not associated with CA Statistics: Mann-Whitney U test, Terpstra-Jonckheere test for trend and Poisson regression


9/29/08 321



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Total 6.1* 3.4

w/o Gaps 4.0* 1.4

Anwar and Shamy 1995 (Egypt)

Reinforced-plastics plant workers (5–22 yr)


NR

328 (145–1,204) 50 (22–92)

Exposure response Duration of exposure, urinary mandelic acid: no significant correlation

All subjects were male; no smokers Controls were admin staff matched for age No information on potential confounding exposures Control samples were collected 1 wk after the exposed samples Cell harvest at 48 h 50 to 100 metaphases/person CA included chromatid gaps, chromatid breaks acentric fragments, and dicentrics. Statistics: Chi-square test

(CA/100 cells) w/o Gaps 3.8* 4.2*

1.7

Lazutka et al. 1999 (Lithuania)

Carpet plant workers (2 mo–21 yr) Plasticware plant workers (2 mo–25 yr)

Exposed carpet 79 plastics 97

Controls 90

NR [0.03–0.32] NR [1–1.4]

NR NR

Exposure response Exposure duration: no association

Controls matched for age Workers at both facilities were exposed to phenol and formaldehyde (higher concentrations than styrene at the carpet plant) Cell harvest at 72 h CA included breaks (chromatid & chromosome) and exchanges; breaks were most common. Gaps not recorded. CA not affected by smoking, age Statistics: t-test on average square root-transformed data, ANOVA

Somorovská et al. 1999 (Bohemia)

Plastics hand-lamination plant workers (14 yr)

Exposed total 44 low 15 medium 12 high 17

Controls 19

[23] (NR) [6] (NR) [13] (NR) [46] (NR)

NR

(CA/100 cells) w/o Gaps 3.3*** 3.3*** 2.5** 3.8***

1.4

Control group from the same factory matched by age but not gender or smoking No exposure to other chemicals Cell harvest at 48 h Smoking habits not related to CA frequency CA defined as chromatid-type and


322 9/29/08



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Exposure response Styrene in air: r = 0.43 (P = 0.001) Blood levels: r = 0.41 (P = 0.001) Exhaled air: r = 0.5, (P < 0.001) Exposure duration: r = 0.55 (P < 0.001)

chromososome-type; gaps scored but not reported CA frequency was higher in females than males Suppression of the proliferative response of T cells to mitogenic stimulations seen in styrene-exposed workers CA also correlated with single-strand breaks Statistics: Mann-Whitney U test, regression analysis

Oberheitmann et al. 2001 (Germany)

Boat building workers (8.7 yr)




NR [<23]

NR

(CA/100 cells) Exchanges 0.22q 0.14

0.25 0.54

0.23 0.10

Controls were from a wood manufacturing training center, not matched for smoking (higher in contols) Cell harvest at 48 h Only reported exchange type CA (analyzed by FISH) Results from X-ray challenge assay indicated that exchange-type aberrations were significantly higher in exposed than historical controls (N = 2) and correlated with lifetime exposure to styrene but not current exposure Statistics: Fisher’s exact (right-sided) and regression analysis

Biró et al. 2002 (Hungary)

Oil refinery workers (NR)


NR

NR

w/o Gaps 3.8 1.8

All individuals were asked to provide information on age, medication, smoking, and drinking habits, and medical and work histories. Exposed included more men (80% vs. 20%) and smokers (80% vs. 20%) than controls; ages were similar Cell harvest at 50 h Smoking did not correlate with CA


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Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Types of aberrations not identified but did not include gaps or aneuploidy Exposed subjects had significant decrease in CD25+ CD4+ cells and an increase in CD45RO+ CD4+ T cells Statistics: Student’s t-test

w/o Gaps 2.3 2.5 2.3 2.0

1.7 3.2

Vodicka et al. 2004a (Czech Republic)

3 Reinforced-plastic lamination plants: (A: 3.4 yr) (B: 5.6 yr) (C: 2.5 yr)

Exposed total 86 plant A 35 plant B 31 plant C 20

Controls plant 16 external 26

[19] (NR) [26] (NR) [11] (NR) [19] (NR)

497 (NR)j 798 (NR) 270 (NR) 308 (NR)

42

No significant differences for chromatid-type aberrations (with or without gaps) or chromosomal breaks. Exposure response No correlation with any marker of styrene exposure

Internal controls: male maintenance workers plant B; External controls: regional hygienic station employees Control groups not matched on age, sex, or smoking but differences considered in the analysis Cell harvest at 48 h Types of CA not completely defined but included chromatid type without gaps, chromatid type with gaps, and chromosome breaks; it is not clear whether total CA without gaps includes CA other than chromatid type without gaps and chromosome breaks CA correlated with age Statistics: Mann-Whitney U test

Vodicka et al. 2004c (Slovak Republic)

3 Groups of tire plant workers (1: 19.4 yrs) (2: 19.1 yrs) (3: 12.1 yrs)

Exposed group 1 53 group 2 41

Controls 16

NR [1.9–3.0] NR (NR)

NR

Total 2.2 1.3*

2.3

All subjects in control group (group 3) and group 1 were male, group 2 included 9 females. Group 1 were workers from the mixing departments and had a higher risk of xenobiotic exposure. Group 2 were workers from production, pressing, fire brigade, and clerks Control group not matched by age or smoking


324 9/29/08



Study populationa


(ppm)b Urinary MA

(mg/g creatin.)


Exposure response Commentsd Confounding factors (such as X-rays, drug use, dietary and life style-factors were recorded in questionnaires Types of CA not defined and it is not clear whether gaps were included. It is also not clear whether data is given as CA/100 cells or % AB.C Workers exposed to 1,3-butadiene, PAHs, and sulfur dioxide Statistics: Mann-Whitney U-test

Total 3.3 3.6

w/o Gaps 2.4 2.5

Migliore et al. 2006b (Italy)

Fiber reinforced-plastics or polyester resin workers from 10 plants (< 1–34 yr)


[8.5 (0.5–123)]

300 (10–1,856)j

Exposure response Chromosomal type CA (without gaps) Airborne styrene: r = 0.393, P = 0.013 MA + PGA: r = 0.293, P = 0.070 4-VPT: r = 0.399, P = 0.012, PHEMA: r = 0.306, P = 0.058 Chromatid type CA Airborne styrene, MA + PGA, 4-VPT – no correlation PHEMA: r = 0.342, P = 0.033

Total population included 95 exposed workers and 98 controls. CA analysis not conducted in all subjects. Controls were from the same geographic area with comparable age. Controls had fewer smokers (42% vs. 53%) but more women (32% vs. 20%) compared with exposed Subjects interviewed for personal, occupational, and medical history (X-rays, viral infections and inflammatory disease, drug use) CA without gaps were higher in smokers but were not related to gender. CAs defined as chromatid and chromosomal type aberrations. Statistics: Multifactorial ANOVA and first-order regression

* P < 0.05, ** P < 0.01, *** P < 0.001. CA = chromosomal aberrations, FISH = fluorescence in situ hybridization, MA = mandelic acid, NR = not reported, PGA = phenylglyoxylic acid, PHEMA = phenylhydroxyethylmercapturic acids, 4-VPT = 4-vinylphenol.


9/29/08 325

a Study population includes both sexes unless otherwise noted. b [Bracketed data were converted from mg/m3 to ppm (1 mg/m3 styrene ≈ 0.23 ppm).] c Types of CA data and units varied but are reported as follows: Total = total of all CA including gaps, w/o Gaps = total CA excluding gaps, % AB.C = % aberrant cells, Breaks = breaks only, Gaps = gaps only, Exchange = exchange type aberrations not otherwise defined. d Potential confounders (e.g., differences in age, sex, smoking, exposures to other chemicals, recent infections, vaccinations, etc.) are noted as identified by the study authors. e Authors only provided CA data for individuals. [Population means were calculated by NTP.] f Range presented for areas of the plant where workers were always present. Concentrations in areas visited for short periods during inspections were below 43 mg/m3 (10 ppm) except on two occasions where concentrations of 91 and 202 mg/m3 (~21and 46 ppm) were recorded. g Concentrations reported in Thiess et al. 1980. h P values not reported. i Marginal increase (0.05 < P < 0.06). j Sum of mandelic and phenylglyoxylic acids. k Calculated sum of chromosome and chromatid breaks. l Results reported for Dean and Clare laboratory. A second laboratory also analyzed a subset of samples collected in 1981. No significant differences reported for either lab. m Units as reported by the study authors, [but considered questionable]. n Calculated values expressed per 100 cells based on 3 gaps in the exposed and 6 gaps in the controls out of 1,100 metaphases examined. o Air concentration was estimated from urine mandelic acid levels. p Average urinary mandelic acid levels were 2.4 mM in laminators that did not use a respirator and 1.3 mM in those who used a respirator. q Study authors did not explain how the value for the total group was less than recorded for either the exposed smokers or exposed nonsmokers.


326 9/29/08

[In general the studies (N = 31) of chromosomal aberrations in styrene-exposed workers

were limited by a small number of subjects and potential confounding from other

workplace exposures.] Most studies included 25 or fewer subjects per group, but some

studies (Andersson et al. 1980, Dolmierski et al. 1983, however there were only 2

controls) (Artuso et al. 1995, Forni et al. 1988, Pohlova and Sram 1985, Somorovská et

al. 1999, Tates et al. 1994, Vodicka et al. 2004c) had somewhat larger populations

(between 30 and 50 in the total exposed or controls) and five studies had populations

between 75 and 100 (Lazutka et al. 1999, Migliore et al. 2006b, Sorsa et al. 1991, van

Sittert and de Jong 1985, Vodicka et al. 2004c).

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

[The exposed and referent populations were usually matched for age, gender, and

smoking habits.] Some studies that did not use matched subjects controlled for variables

(such as age, gender, and smoking habits) in the analysis or reported that age, smoking,

and gender distribution were similar between groups. Studies that did not meet these

criteria include Thiess et al. (1980) (although the authors stated that smoking information

was recorded), Sorsa et al. (1991) [not clear whether smoking and age were controlled for

in the dose-response regression analysis], Biró et al (2002) (smoking and gender differed

between exposed and controls but ages were similar), Oberheitmann et al. (2001) (similar

ages but smoking differed between exposed and controls) and Vodicka et al. (2004c).

[None of these studies found an association between chromosomal aberrations and

styrene exposure, see below.] Several studies evaluated the effects of potential

confounders such as smoking, age, and gender on aberration frequency. Of the 13 studies

that evaluated smoking, 11 (Andersson et al. 1980, Artuso et al. 1995, Biró et al. 2002,

Camurri et al. 1983, 1984 [considered as one study], Lazutka et al. 1999, Maki-

Paakkanen 1987, Meretoja et al. 1978a, Pohlova and Sram 1985, Somorovská et al. 1999,

Tates et al. 1994, Tomanin et al. 1992) reported that smoking did not affect, or was not

correlated with an increase in chromosomal aberrations; one study (Maki-Paakkanen et

al. 1991) found higher chromosomal aberrations in smokers in the control group than

non-smoking controls, and another study (Sorsa et al. 1991) reported a positive

correlation between chromosomal aberration frequency and smoking. Conflicting results

were found for the two studies that evaluated age; Sorsa et al. reported that age was

correlated with chromosomal aberration frequency; however, Lazutka et al. did not find


9/29/08 327

an effect. One study (Somorovská et al. 1999) reported that chromosomal aberration

frequency was higher in females compared with males; however, Pohlová and Srám did

not find any differences related to gender.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

[Common study quality issues, which are related to the measurement of chromosomal

aberrations included cells cultured too long for the peak period of chromosomal

aberrations, inadequate number of metaphases scored per individual, or incomplete

exposure data. These potential quality issues are identified in the “Comments” column in

Table 5-13. Most of the studies examined at least 100 metaphases per person; however,

current guidelines recommend a minimum of 200 metaphases per subject (Albertini et al.

2000). Studies that scored fewer than 100 metaphases per person are identified in the

“Comments” column. Several studies used cell-culture times that were longer than ideal

(i.e., comprising mostly second division cells). In some cases the authors stated that the

longer culture times were chosen so that a larger number of mitotic cells would be

available for scoring.]

[It is difficult to compare results across studies because the studies were not consistent in

data reporting]. Some studies reported the percentage of cells per subject with

chromosomal aberrations, while others reported the mean number of aberrations per 100

cells. Studies also varied in the type and description of the aberrations reported; some

only gave total estimates, whereas others reported the frequency of specific types of

chromosomal aberrations (e.g., gaps, breaks, exchanges) or general categories of

chromosomal aberrations (e.g., with or without gaps). The data in Table 5-13 include the

most comprehensive measures of chromosomal aberrations reported by the study authors

and are identified in the “Results” column. When available, information is presented for

total chromosomal aberrations without gaps.

One study (Oberheitmann et al. 2001) measured exchange-type chromosomal aberrations

and did not find a significant increase in styrene-exposed workers compared with

controls. [The study was limited by small numbers of subjects and unmatched controls

(wood manufacturing industry) for smoking (more controls than exposed smoked).]

However, after X-ray challenge, the rate of exchange type aberration frequency was


328 9/29/08

higher in exposed compared with historical controls (N = 2). The response was correlated

with cumulative lifetime exposure to styrene but not current exposure (see Section

5.4.4.2).

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Of the remaining 30 studies, 17 studies ― Meretoja et al. (1978a, 1977), Fleig and Thiess

(1978) (high exposure subgroup), Högstedt et al. (1979), Andersson et al. (1980),

Dolmierski et al. (1983), Camurri et al. (1983, 1984), Hansteen et al. (1984), Pohlová

and Srám (1985) (only for one of two plants, and only for one of two samplings), Forni et

al. (1988), Mäki-Paakkanen et al. (1991) (nonsmokers only), Tomanin et al. (1992) (high

exposure factory), Tates et al. (1994), Artuso et al. (1995), Anwar and Shamy (1995),

Lazutka et al. (1999), and Somorovska et al. (1999) ― reported a significant increase in

the frequency of chromosomal aberrations in the exposed population (or subgroup of

exposed workers) compared with the controls. [The studies were not consistent in the

types of aberrations found to be elevated (see Table 5-13).] Three of these studies found

significant increases in gaps only (Dolmierski et al. 1983, Hansteen et al. 1984, Pohlova

and Sram 1985). [Findings reported by Dolmierski et al. were limited by the small

numbers of controls (N = 2), and findings in some other studies were limited by potential

confounding from other occupational exposures.] Workers in the study reported by Tates

et al. were also exposed to dichloromethane. A positive correlation between styrene

exposure (TWA) and chromosomal aberrations was found in one of two exposed

subgroups but not the pooled population; however, positive correlations were also found

between dichloromethane exposure (TWA) and chromosomal aberrations in that

subgroup as well as the total exposed subjects. Workers in the study reported by Lazutka

et al. were exposed to higher levels of phenol and formaldehyde than styrene, [but no

dose-response analysis was performed]. The authors stated that the literature on

chromosome damage by occupational exposure to formaldehyde is not consistent and no

literature was available on the genotoxic effects of environmental exposure to phenol.

Workers in other studies were often exposed to other agents such as peroxides, methylene

chloride, and acetone.

[In general, “positive” results were observed in studies with higher levels of exposure or

in the high-exposure subgroup. An exception is the Lazutka et al. study (exposure


9/29/08 329

between 0.03 and 1.4 ppm). This study was also one of the larger studies, and as

mentioned above the workers were also exposed to formaldehyde and phenol.] Migliore

et al. did not report an increase in chromosomal aberration frequency in the exposed

workers versus the controls, but did find positive correlations with chromosomal type

aberrations (without gaps) with various measures of styrene exposure (styrene air levels

and urinary metabolites, and chromatid type aberrations with urinary

phenylhydroxyethylmercapturic acids. The average exposure levels were low in this

study. In addition to Migliore et al., positive correlations with measures of styrene

exposure were also reported by Camurri et al. (1983, but not 1984 analysis), Andersson

et al. (cumulative styrene exposure, increase observed in both low and high subgroup but

only significant in the low group), Tates et al. (in one subgroup but not in the total

population), and Somorovska (air, exhaled air, urinary metabolites, and blood levels).

Artuso et al. used a multivariate regression model and found a higher RR for

chromosomal aberrations for high styrene exposure (RR = 1.71, 95% CI = 1.25 to 2.33)

than low styrene exposure (RR = 1.38, 95% CI = 0.98 to 1.94). (Smoking, alcohol

drinking, and diagnostic X-rays were not risk factors for chromosomal aberrations in this

model). Fleig and Thiess, and Tomanin et al. reported higher chromosomal aberrations in

the high-exposure subgroup (as assessed by air and urinary metabolite levels) compared

with the low-exposure subgroup. Pohlová and Srám measured urinary metabolites and

chromosomal aberrations in the same workers (at two different plants) at two different

sampling times. Urinary metabolites (see Table 5-13) and chromosomal aberrations

increased at the second sampling. At the second sampling, styrene-exposed workers in

Plant A had a significant increase (P < 0.01) in rates of gaps (from 1st sampling to 2nd

sampling), and styrene-exposed workers in Plant B had significantly higher rate of

percent aberrant cells (cells with breaks or exchanges).

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

In contrast, the following studies did not find a positive correlation between styrene

exposure (either air levels or urinary metabolite level) and chromosomal aberrations:

Watanabe et al. (1983), Sorsa et al. (1991), Anwar and Shamy (1995), and Vodicka et al.

(2004a). Several studies did not report a significant correlation with exposure duration

(Anwar and Shamy 1995, Hagmar et al. 1989, Lazutka et al. 1999, Tomanin et al. 1992,

Watanabe et al. 1981), while Somorovska et al. (1999) and Mäki-Paakkanen et al. (1991)


330 9/29/08

did report a significant correlation with exposure duration. [The ability to detect dose-

response relationships is limited by small numbers in most studies; studies with the larger

numbers of exposed subjects include Migliore et al. (2006b), Somorovska et al. (1999),

Sorsa et al. (1991), and Vodicka et al. (2004c).]

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Studies that did not find any significant increases in chromosomal aberrations in workers

exposed to styrene include Thiess et al. (1980), Watanabe et al. (1983, 1981), Nordenson

and Beckman (1984), Mäki-Paakkanen (1987), Jablonicka et al. (1988), Sorsa et al.

(1991), Biró et al. (2002), and Vodicka et al. (2004c, 2004a). van Sittert and de Jong

(1985) used pre-exposure measures for the reference group and followed the study

population for a couple of years. An increase in chromosomal aberrations was observed

for only 1 of the 3 follow-up years. The authors reported that there were no changes in

styrene, propylene oxide, and benzene air levels from 1978 to 1981, thus, they did not

think the increase in chromosomal aberrations in 1980 was due to occupational exposure,

and Migliore et al. (2006b) reported a positive dose-response relationship but no

significant pair-wise comparison, [which complicates the classification (in terms of

positive or negative) of these studies. The study by Sorsa et al. had limited

documentation on its study population.] Mäki-Paakkanen et al. (1991) reported an

increase in chromosomal aberration frequency in non-smokers but not in smokers or the

total population. Watanabe et al. (1983) reported that there was a marginal (0.05 < P <

0.06) increase in chromosomal aberration frequency in the exposed group compared with

the controls. [Most of the “negative” studies had somewhat lower levels of exposures

except for Watanabe et al. (1983, 1981) and Jablonicka et al. (1988).]

As mentioned above, there have been several reviews or evaluations of the cytogenetic

effects of styrene. Early reviews such as IARC (1994a,b) and Scott and Preston (1994a)

are not discussed here since they only include a subset of the available literature to date.

Bonassi et al. (1996) performed a meta-analysis of 25 (22 with results for chromosomal

aberrations) biomonitoring studies of occupational exposure to styrene. The review

included all studies up to Artuso et al. (in Table 5-13), but did not include two earlier

studies, Dolmierski et al. (1983) and van Sittert and de Jong (1985). The authors found a

positive association (weighted frequency ratio = 2.18, 95% CI = 1.52 to 3.13, weight was


9/29/08 331

assigned to each study depending on its sample variance) between styrene exposure level


greater or less than a threshold value of 30 ppm for an 8-hour time-weighted average

(which was the median exposed-group exposure level in the identified studies).

1

2

3

4

5

6

7

8

9

10

11

12

13

15

16

17

18

19

20

21

22

23

24

25

26

27

28

Cohen et al. (2002) concurred with the Bonassi et al. review and also noted that the

finding of dose-response relationships makes confounding unlikely. They concluded that

there was “compelling evidence” of a positive association between styrene exposure at

occupational levels and the frequency of chromosomal abnormalities. However, a review

by Henderson and Speit (2005) concluded that the evidence for chromosomal aberrations

was conflicting. This review included 27 studies [it did not include Dolmierski et al.

(1983), van Sittert and de Jong (1985), Vodicka et al. (2004c), and Migiliore et

al.(2006a) The authors stated that the Bonassi review did not account for study quality or

the type of chromosomal aberrations (such as including gaps).

Micronucleus formation 14 Details on the study population, exposure levels, study design, and results for structural

micronuceus formation are summarized in Table 5-14, and the findings are discussed

after the tables.

The current guidelines for investigating the frequency of micronucleated blood

lymphocytes or epithelial cells in humans are presented by Albertini et al. (2000). The

cytokinesis-block micronucleus technique is the method of choice [first used in studies on

styrene exposure by Mäki-Paakkanen et al. 1991]. Cytochalasin B (Cyt-B) is added to the

cell culture to block cells from dividing after they have undergone one round of

replicative synthesis since mitogen stimulation; such cells are binucleate. Most studies

score 1,000 to 2,000 binucleated cells per subject. The results are expressed as the

number of micronucleated cells per 1,000 binucleate cells or the percentage of cells with

micronuclei (Table 5-14). Some studies also indicated the number of kinetochore-

positive/centromere-positive micronuclei and the number of kinetochore-

negative/centromere-negative micronuclei.


Table 5-14. Micronuclei in lymphocytes from workers occupationally exposed to styrene Styrene exposure

Mean (range)


Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)

Results (mean ± SD)

Exposure response Commentsc

Meretoja et al. 1977 (Finland)

Polyester plastic manufacturing workers from 3 plants (0.6–8.5 yr)


NR

[721 (23–3,257)]

MN/1,000 cells [8.8 ± 2.9]d [0.8 ± 1.1]

All subjects male; unmatched controls selected from outside the factory environment but similar age ranges No exposure to known clastogenic agents or recent viral infections or vaccinations Mainly 2nd division cells scored Statistics: not performed

Hogstedt et al. 1983 (Sweden)

Fiberglass-reinforced polyester resin manufacturing workers (1–23 yr)

Preserved cytoplasm exposed 38 controls 20

Hypotonic treatment exposed 38 controls 20

13 (1–36)

65 (9–316)

MN/1,000 cells 5.9** 3.6

4.3 3.7

Exposure response Styrene air concentrations, exposure duration, cumulative dose, and urinary mandelic acid: no correlation

All subjects male controls from nearby mechanical industry groups and matched for age Workers interviewed about potential confounders including occupational and medical history, viral infections, drug use, smoking and alcohol habits and exposure to X-rays and heavy metals. The following differences were found: (1) smokers– exposed 45%, controls 40%; (2) X-rays– exposed 29%, controls 25%; (3) drug use– exposed 11%, controls 15% Workers exposed to phthalic acid anhydride, maleic acid anhydride, propylene and/or ethylene glycol, hydroquinone, methylethyl ketone peroxide, cobalt salt, methylene chloride, solvents, and acetone MN analyzed by 2 methods: (1) preserved cytoplasm, and (2) hypotonic treatment with KCl Statistics: (1) Effect of exposure: multiple regression analysis controlling for smoking and

332 9/29/08


9/29/08 333



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



age using log-transformed values, (2) exposure response– Spearman rank correlation coefficient

Nordenson and Beckman 1984 (Sweden)

Glass-fiber reinforced polyester plant workers (1–26 yr)




24 (NR)

(mM/L) NR (< 2)

MN/1,000 cells 3.5* 0.8

3.3* 1.0

3.6* 0.7

All subjects male controls were office workers Exposed to acetone No indication if slides were coded Cultures from 4 subjects (3 exposed and 1 control) were unsuitable for analysis MN similar in smokers and non-smokers Exposed also had significantly higher number of subjects with > 3 MN/1,000 cells (P < 0.001) Statistics: one-tailed exact test






[23 (8–60)]

(mM/L) 2.0 (0–7.3)

% cells with MN 1.5 ± 0.1e 1.6 ± 0.1

1.4 ± 0.2 1.6 ± 0.1

1.6 ± 0.2 1.6 ± 0.3

Exposure response Duration, air, or urinary MGA: no correlation

Controls were office workers matched by sex and smoking. No differences between groups in alcohol and drug intake, vaccinations, recent viral infections, or previous exposure to chemicals Statistics: t-test (one sided); analysis for exposure response not reported

Hagmar et al. 1989

Glass reinforced polyester plastic

PHA exposed 20

[13 (0.9–127)]

NR

MN/1,000 cells 4.3

All but one subject (exposed group) were male Some subjects reported recent exposures to X-rays


334 9/29/08



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



(Sweden)

workers (0.1–25.4 yr)

controls 22

PWN exposed 20 controls 22

4.4

5.9 7.0

Exposure response Employment length: no association

(65% exposed, 55% controls) and regular prescription drug use (15% exposed, 23% controls). More smokers in controls (50%) than exposed workers (30%) Workers exposed to low levels of acetone and methylene chloride Parallel cultures were set up and stimulated with pokeweed (PWN) or phytohemagglutinin (PHA) MN frequency (PHA) was correlated with age Statistics: significant testing of difference in means, after average square root transformation, adjusted for age and smoking; multiple linear regression evaluating employment length, smoking, and age

Brenner et al. 1991 United States

Reinforced-fiberglass plastic boat workers (2.7 yr)


Controls 9

11.2 (1–44) 27.2 (7–44 6.8 (1–18)

[24.3 (9.6–250)] [52 (9.6–250)] [18 (9.6–50)]

MN/1,000 cells 10.3 ± 0.4** 10.8 ± 0.6 10.0 ± 0.5

6.5 ± 0.5 Exposure response Positive association by ANOVA, no response observed with continuous variables (air, urinary markers, and cumulative exposure)

All exposed subjects were male; controls included male and female library workers Controls were library workers at a university and differed by sex and current smoking (which were retained in the analysis), education, and medication (which could not be retained in the analysis due to small numbers of subjects). No differences with respect to age, caffeine and alcohol intake, recency of colds or X-rays, other tobacco-related exposures, and exposure to wood smoke or solvents Co-exposure to acetone and methylene chloride Gender, education, and smoking had no effect on MN when analyzed by ANOVA


9/29/08 335



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



Statistics: log transformed, Wilcoxon rank-sum test, ANOVA, which included gender, smoking, exposure, and educational status, used to evaluate exposure response in 3 exposure groups


Reinforced plastic workers (smokers – 6.4 yr, non-smokers – 7.2 yr)




[~ 70] (NR)f

(mM) 9.4 (< 1–21.5)

11.0 (< 1–16.6)

6.5 (< 1–21.5)

% cells with MN 1.4 ± 0.6 1.2 ± 0.8

1.3 ± 0.7 1.1 ± 0.8

1.5 ± 0.4 1.4 ± 0.8

Controls from a research institute Age, sex, smoking status, health status, alcohol and drug intake, viral infections, vaccinations, and exposure to other chemicals were considered First study to use cytokinesis-block technique 500 binucleated cells/subject analyzed Statistics: Chi square test


Reinforced-plastics industry workers from 32 enterprises (NR)

Past exp. (index pts) Laminators low 15 high 13


Controls other factory 31 plastics factory 6

All subjects exposed 50 controls 37

43 (5–182)

11 (1–133)

(mM) 2.2 (NR)g,h

% cells with MN 0.6 ± 0.5 0.7 ± 0.4

0.4 ± 0.3 1.0 ± 0.2

0.8 ± 0.5 0.8 ± 0.3

NR NR

Exposure response

Total population included 248 exposed workers, including 154 laminators, and 63 controls. MN results available on a subset; past-exposure index not available on all subjects Exposed groups divided into 2 groups based on past-exposure index points derived from exposure duration and concentrations, urinary metabolites, and job type Cytokinesis-block technique 500 binucleated cells/subject analyzed MN was associated with age (P = 0.03), but not with smoking Statistics: regression analysis including exposure, smoking and age, details not reported


336 9/29/08



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



No association with styrene exposure

Tomanin et al. 1992 (Italy)

Polyester resin workers at 2 factories: fiberglass tanks (1–18 yr) or fiberglass boats (1.5–15 yr)

Factory 1 exposed 7 controls 7

Factory 2 exposed 12 controls 12

NR [4.8–23]

NR [26–100]

186 (46–345)

725 (423–1,325)

% cells with MN 8.7 ± 4.0 10.2 ± 4.4

12.6 ± 6.6 8.5 ± 3.3

Exposure response Exposure duration: no correlation Urinary MA: weak correlation R = 0.61 (P value not given)

Controls matched for sex, age, and smoking Subjects questioned about previous exposure to genotoxins, smoking and alcohol habits, recent viral infections or vaccinations, and exposure to X-rays Cytokinesis-block technique Different number of cells scored in exposed and control groups No significant effect with smoking Statistics: Mann-Whitney U test (2-sided), and simple linear regression

Yager et al. 1993 (United States)

Boat manufacturing workers (0.5–27 yr)

Exposed 48

[15 (0.2–54)]

NR

MN/1,000 cells 8.9 ± 0.9e

Exposure response No association with exposure to styrene after adjusting for gender

No controls, exposed subjects 54% male, 46% female. Longitudinal study; exposure measured by personal monitors and concentrations in exhaled breath 7 times over a 1-yr period Cytokinesis-block technique MN frequency increased with age and was higher in females Statistics: linear regression analysis including styrene exposure, age, sex, lifestyle variables (such as smoking, alcohol intake, some dietary factors, drug intake, immunizations, infections, exposures from hobbies and home repairs) and occupational history


9/29/08 337



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)






Controls 23

[16 (0–138)] [20 (0–138)] [12 (0–34)]

NR

MN/1,000 cells 35.1 ± 20*** 32.3 ± 24*** 38.2 ± 13***

14.3 ± 7.3

Exposure response Duration: positive correlation (P = 0.001) in Group 2 only Concentration: Group 1 only (P = 0.05) Duration × TWA: P = 0.035 for styrene/DCM exposure

Controls matched for age, sex, and smoking Workers divided into 2 groups with similar working conditions, but blood samples were taken 1 wk apart Subjects questioned about health status, exposure to X-rays, drug use, and smoking and alcohol habits; blood samples tested for some viral infections Workers exposed to dichloromethane (genotoxin) Cytokinesis-block technique No significant differences for MN between smokers and nonsmokers Significant difference in MN between the 2 exposure groups (P = 0.04) Statistics: one-tailed Mann-Whitney U test and bivariate regression analysis

Van Hummelen et al. 1994 (Belgium)

Fiberglass-reinforced plastic pipes and cisterns workers (2.9 yr)



[7 (0.5–25)]

102 (11–649)

MN/1,000 cells 3.28 ± 0.28e 4.32 ± 0.55

3.50 ± 0.34 4.75 ± 0.71

Exposure response Air, urinary MA no correlation

Study consisted of 52 exposed and 24 non-nonexposed workers, but cytogenetic results were not available on all subjects because of technical problems All subjects were males, controls were from a different factory (pallet production and repair) Subjects were interviewed regarding exposure to potential carcinogens and mutagens, smoking habits, diet, viral infections, vaccinations, chemotherapy, and X-rays. Exposed were significantly older (31 vs. 27), consumed less


338 9/29/08



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



alcohol (1 vs 2.6 drinks/d), and had more recent X-rays (0.6 vs. 3.6 yr ago) Cytokinesis-block technique No correlation of MN with age, smoking, medical history, or other lifestyle factors Statistics: two-tailed Mann-Whitney U test, Spearman ranked correlation, ANOVA (using square root of value)

Anwar and Shamy 1995 (Egypt)

Reinforced-plastics plant workers (10–22 yr)


NR

328 (145–1,204)

50 (22–92)

MN/1,000 cells 6.55 ± 3.47 6.00 ± [2.83] Exposure response Duration, urinary MA: no correlation

All subjects were male; controls were administrative staff from the same factory, matched for age, and not exposed to genotoxic agents. No smokers were included Exposure measurements were done on the “pilot study” that included 70 exposed workers and 68 controls No information on potential confounding exposures Control samples were collected 1 wk after the exposed samples Cytokinesis-block technique MN did not correlate with age or urinary thioester (UT) levels (biomarkers of electrophilic compounds). UT levels significantly higher in exposed than controls (pilot study) 500–1,000 binucleated cells/individual Statistical analysis: Chi-square test

Holz et al. Styrene Total % cells with MN Controls matched for age and sex and from the


9/29/08 339



Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



1995 Germany

production plant workers (1–34 yr)

exposed 25 controls 25




NR [0.02–> 0.9] NR [≤ 0.01]

13.3–43.9 (NR)i 4.3–5.5 (NR)

13.3–43.9 (NR)i

4.3–5.5 (NR)

10–49.4 (NR) 5.6–6.3 (NR)

20.3–32.4 (NR) 2.8–4.6 (NR)

1.90 ± 0.78 1.87 ± 0.71

% K+ MN 39.4 ± 10.2** 31.8 ± 8.2

38.3 ± 11.3* 30.3 ± 7.9

42.1 ± 7.4* 33.3 ± 8.51

same facility Subjects questioned about alcohol consumption, smoking, drug use, and exposure to aromatic hydrocarbons outside the workplace Workers exposed to aromatic hydrocarbons: ethylbenzene (highest exposure), benzene, toluene, and xylene Modified cytokinesis-block methodology CREST staining to detect kinetochore-positive (K+) MN No confounding effect from smoking K+ MN indicates aneuploidy. Authors suggested that increase in K+ MN was consistent with exposure to benzene Statistical analysis: t-test

Karakaya et al. 1997 (Turkey)

furniture workers (10 yr)




30 (20–300)

207 (14–1,482)g

12 (0–38)g

% cells with MN 1.98 ± 0.50 2.09 ± 0.35

1.91 ± 0.46 2.20 ± 0.31

2.18 ± 0.57 1.82 ± 0.30 Exposure response Duration: nonsignificant trend

All subjects were male; controls were university employees matched on age and smoking Subjects interviewed about occupational, family, and dietary history No information on other current workplace exposures MN in control smokers were significantly higher than control non-smokers, and was higher in older subjects (> 36 yr) in both controls and exposed groups Urinary thioethers were significantly higher in exposed than controls but did not correlate with MN in the exposed group




Study populationa

(yrs employed)

Number of subjects

Air (ppm)b

Results Urinary mandelic acid

(mg/g creatinine)

(mean ± SD) Exposure response Commentsc

Statistics: t-test, Spearman rank correlation, average square root transformation

Laffon et al. 2002a Spain

Fiberglass-reinforced-plastics production workers (≥ 7 yr)


< 20 (NR)e

313–353 (NR)j

MN/1,000 cells 24.6 ± 1.5**e 13.9 ± 0.81 Exposure response Duration: positive correlation (P < 0.001)

All subjects were male; controls were university employees Subjects interviewed on smoking, alcohol consumption, medication, recent viral infections, vaccinations, X-rays, and previous occupational exposure to chemicals More controls smoked (63%) than exposed (36%), but exposed subjects had smoked longer Workers also exposed to peroxides Cytokinesis-block technique MN non-significant increase with age, but significant increase with smoking (# of cigarettes and years smoked) in exposed group Statistics: ANOVA (one way), Student’s t-test, Pearson correlation

Teixeira et al. 2004 NR

2 small reinforced-plastics plants (12 yr)


Men exposed 18 controls 18

Women exposed 10 controls 10

27 (2–91)

401 (47–1,490)g

MN/1,000 cells 3.68 ± 0.46e 2.82 ± 0.47

2.50 ± 0.35 2.00 ± 0.33

5.80 ± 0.77 4.30 ± 1.03

Controls were office workers and were similar in age, sex ratio, and smoking habits as exposed group Subjects queried about lifestyle factors (smoking and alcohol habits, medications, X-rays, and diet), and occupational exposures to chemicals Workers also exposed to low levels of toluene and acetone (< 1% of styrene levels) Cytokinesis-block technique MN significantly (P < 0.05) higher in styrene-

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Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)


exposed women than men, smoking not significantly related to MN Statistics: Student’s t-test

Godderis et al. 2004 Belgium

Fiberglass-reinforced plastic workers from 2 plants (7 mo–38 yr)

Lymphocytes MNBC 38 controls 41

MNMC 38 controls 41

Nasal cells exposed 23 controls 17

9.5 (0–36.6)f

9.5 (0–36.6)

7 (0–24.6)

202 (ND–618)

202 (ND–618)

162 (ND–104)

MN/1,000 cells 3.93 ± 2.8* 2.65 ± 1.94

0.71 ± 0.9*** 0.11 ± 0.20

0.52 ± 0.49* 0.23 ± 0.31 Exposure response Employment duration: positive association with MNBC and MNMC Average styrene exposure: positive association with nasal cell MN

All subjects were male; controls recruited from electrical wire and cable manufacturing companies from the same region and matched on age and smoking. Controls and exposed had similar alcohol intake, blood lead levels. 3 exposed and 7 controls had urinary chromium levels above the reference value but under ACGIH biological exposure index Cytokinesis-block technique Binucleated (MNBC) and mononucleated (MNMC) lymphocytes and nasal epithelial cells analyzed MNBC correlated with age (P = 0.014) in the total population, MNBC and MNMC correlated with smoking (P < 0.05) in the exposed groups and XRCC1 polymorphism (P < 0.05) in the total population Statistics: Mann-Whitney U test for lymphocytes and unpaired t-test for nasal cells, bivariate correlation analysis multiple regression analysis (backward, stepwise)

Vodicka et al. 2004a (Czech Republic)

3 Reinforced-plastic lamination plants (A: 3.4 yr)

Exposed total 86 plant A 35 plant B 31

[19] (NR) [26] (NR) [11] (NR) [19] (NR)

497 (NR)g 798 (NR) 270 (NR)

% cells with MN 15.1 ± 6.7 17.9 ± 8.1**k 13.4 ± 4.3

Internal controls: male maintenance workers External controls: employees of the regional hygienic station Controls were older (+8.7 yr), had fewer men

9/29/08 341




Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)


(B: 5.6 yr) (C: 2.5 yr)

plant C 20

Controls plant 16 external 26

308 (NR)

42

12.5 ± 4.7

11.6 ± 4.9 15.6 ± 6.9 Exposure response (correlations with styrene exposure) Air levels: P < 0.001 Blood levels: P < 0.001 Cumulative exposure P < 0.05

(52% vs. 71%), and fewer smokers (19% vs. 51%) than exposed but had a similar socioeconomic background. Differences accounted for in the analysis Cytokinesis-block technique MN significantly increased with age, were higher in women and external controls Statistical analysis: Mann-Whitney U test, Spearman correlation analysis

Migliore et al. 2006b (Tuscany and Parma, Italy)

Fiberglass reinforced-plastics workers from 13 plants (1–34 yr)




[8.5 (0.5–123)]

300 (10–1856)f

% cells with MN 13.8 ± 0.5***e 9.2 ± 0.42

% cells C+MN 7.43 ± 0.34*** 4.75 ± 0.37 % cells C–MN 5.76 ± 0.3*** 3.20 ± 0.3 Exposure response Air levels: No correlation with total

Controls were from the same geographic area with comparable age. Controls had fewer smokers (42% vs. 53%) but more women (32% vs. 20%) compared with exposed Subjects interviewed for personal, occupational, and medical history (X-rays, viral infections and inflammatory disease, drug use) 4-Vinylphenol conjugate levels were available on the Parma workers (26 males and 19 females) MN measured by FISH, centromere + (C+) and centromere – (C–) cells also scored (2 scorers used) Smoking had no effect on total MN and C+MN frequency but was associated with a decreased C–

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Study populationa

(yrs employed)

Number of subjects

Air (ppm)b


(mg/g creatinine)



MN: C+ or C–MN MA+PGA metabolites: Total MN and C–MN, P > 0.05, C+MN, P = 0.011 4-VPT metabolites: total MN and C+MN, P < 0.01

MN frequency; MN and C+MN increased with age, and MN (total) were higher in females than males MN also higher in GSTT1-null exposed subjects Statistics: Multifactorial ANOVA including smoking habits, age, and sex MN data on a subset of male workers from the Tuscany cohort (42 exposed workers and 25 controls was reported by Miglore et al. 2006a. The exposed in this subset also had increased MN (13.8) compared with the controls (6.2)

* P < 0.05, ** P < 0.01, *** P < 0.001. FISH = fluorescence in situ hybridization, MN = micronuclei, NR = not reported. a Study population includes both sexes unless otherwise noted. b [Bracketed data were converted from mg/m3 to ppm (1 mg/m3 styrene ≈ 0.23 ppm).] c Potential confounders (e.g., differences in age, sex, smoking, exposures to other chemicals, recent infections, vaccinations, etc.) are noted as identified by the study authors. d No P value provided but reported as an increase by study authors and identified as a positive study by Scott and Preston (1994a). e Mean ± SE. f Air concentration was estimated from urine mandelic acid levels. g Sum of mandelic and phenylglyoxylic acids. h Average urinary mandelic acid levels were 2.4 mM in laminators that did not use a respirator and 1.3 mM in those who used a respirator. i Values are the range of means reported before and after work shift. j Range of means from three samplings. k Compared with plant controls.


344 9/29/08/08

Micronuclei were measured in peripheral blood lymphocytes from workers exposed to

styrene in 20 studies and in nasal epithelial cells in one study (Table 5-14). All but three

studies (Anwar and Shamy 1995, Maki-Paakkanen et al. 1991, Sorsa et al. 1991) scored a

minimum of 1,000 cells per subject. [As with the chromosomal aberration studies, data

quality issues (e.g., small number of subjects, unmatched controls, and exposure to other

clastogenic agents) were identified for several of the studies. About half of the studies

included fewer than 25 subjects per group. Most of the studies included control groups

matched on one or more of the following variables: age, gender, or smoking. Studies that

did not report using matched control groups included Meretoja et al. (1977), Hagmar et

al. (1989), Brenner et al. (1991), Sorsa et al. (1991), and Vodicka et al. (2004a). Most of

the studies that did not use matched subjects controlled for variables (such as age, gender,

and smoking habits) in the analysis or reported that age, smoking, and gender distribution

were similar between groups. Only one study (Sorsa et al. 1991) did not appear to meet

that criterion, although it was not clear whether smoking and age were controlled for in

the dose-response regression analysis. ]

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

[Most studies evaluated the effects of potential confounders such as smoking, age, and

gender on micronuclei, but the results were mixed]. Only three studies (Godderis et al.

2004, Laffon et al. 2002a, Migliore et al. 2006b) reported that smoking was correlated

with an increase in micronuclei, but nine studies (Brenner et al. 1991, Hagmar et al.

1989, Holz et al. 1995, Nordenson and Beckman 1984, Sorsa et al. 1991, Tates et al.

1994, Teixeira et al. 2004, Tomanin et al. 1992, Van Hummelen et al. 1994) did not

show a correlation with smoking. Migliore et al. (2006b) reported that smoking was not

correlated with total micronuclei but was correlated with a decrease in centromere-

negative micronuclei. Six studies reported a significant correlation with age (Godderis et

al. 2004, Hagmar et al. 1989, Migliore et al. 2006b, Sorsa et al. 1991, Vodicka et al.

2004a, Yager et al. 1993), but four studies reported no correlation with age (Anwar and

Shamy 1995, Brenner et al. 1991, Laffon et al. 2002a, Van Hummelen et al. 1994)

[although a non-significant increase with age was reported by Laffon et al.]. Four studies

(Migliore et al. 2006b, Teixeira et al. 2004, Vodicka et al. 2004a, Yager et al. 1993)

reported that micronuclei were higher in females compared with males. However,

Brenner et al. (1991) did not find any differences related to gender.


9/29/08 345

Yager et al. (1993) conducted a longitudinal study (without controls) that compared

styrene exposure (measured at several times during a one year period) with micronucleus

frequency. No correlation was found between styrene exposure (air levels and exhaled

air) and micronucleus frequency.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Micronuclei were significantly increased in styrene-exposed workers in at least one

exposed group in 10 (of the remaining 19) studies (Brenner et al. 1991, Godderis et al.

2004, Hogstedt et al. 1983, Holz et al. 1995, Laffon et al. 2002a, Meretoja et al. 1977,

Migliore et al. 2006b, Nordenson and Beckman 1984, Tates et al. 1994, Vodicka et al.

2004a). Only kinetochore-positive micronuclei [an indicator of aneuploidy] were

increased in the study by Holz et al. (1995), and Vodicka et al. (2004a) reported

significantly more micronuclei in a single subgroup of workers (one of three plants). Of

the 12 studies that evaluated dose-response relationships, five reported a significant

correlation with styrene exposure (Brenner et al. 1991, Tates et al. 1994 (in one of the

subgroups but not the pooled population), Laffon et al. 2002a, Godderis et al. 2004, and

Vodicka et al. 2004a). Workers in the study reported by Tates et al. were also exposed to

dichloromethane; however, no correlation was found between dichloromethane [which is

a genotoxin] and micronuclei. Holz et al. (1995) attributed the increase in kinetochore-

positive micronuclei to exposure to benzene. Workers in other studies were also exposed

to other chemicals such as peroxides, organic solvents, acetone (Brenner et al. 1991,

Hogstedt et al. 1983, Laffon et al. 2002a, Nordenson and Beckman 1984), but it was not

reported whether these chemicals can cause micronuclei.

Nine studies did not find increased micronuclei frequency in the exposed groups (Anwar

and Shamy 1995, Hagmar et al. 1989, Karakaya et al. 1997, Maki-Paakkanen 1987,

Maki-Paakkanen et al. 1991, Sorsa et al. 1991, Teixeira et al. 2004, Tomanin et al. 1992,

Van Hummelen et al. 1994). The exposed populations in these studies ranged from 7 to

50 subjects, and the reported mean styrene concentrations ranged from about 7 to 70

ppm, which were similar to those reported for the positive studies. Bonassi et al. (1996)

conducted a meta-analysis of 10 studies of micronucleus frequency in styrene-exposed

workers (Brenner et al. 1991, Hagmar et al. 1989, Hogstedt et al. 1983, Maki-Paakkanen

1987, Maki-Paakkanen et al. 1991, Meretoja et al. 1977, Nordenson and Beckman 1984,


346 9/29/08/08

Sorsa et al. 1991, Tates et al. 1994, Tomanin et al. 1992) and concluded that the data

were inconclusive. Of the 10 studies published since that analysis, five reported positive

associations. Cohen et al. (2002), noting a general lack of evidence of a significant dose

response and inadequate control for potential confounders, concluded that there was no

compelling evidence in humans that exposure to styrene was associated with

micronucleus formation.

1

2

3

4

5

6

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Sister chromatid exchange 7 Details on the study population, exposure levels, study design, and results for structural

sister chromatid exchange (SCE) are summarized in Table 5-15, and the findings are

discussed after the tables.

SCE scoring is conducted in second-division metaphases and requires DNA replication in

the presence of bromodeoxyuridine (BrdU) for two consecutive cell cycles, or at least the

first of two consecutive cell cycles (Albertini et al. 2000). It is necessary to score 30 to 50

second-division metaphase cells to obtain a stable estimate of the mean; however, a

minimum of 80 cells is recommended to identify a small proportion (~10%) of high-

frequency SCE cells (cells with an abnormally high number of SCEs). Seven studies

included subjects that had fewer than 30 metaphases scored (Andersson et al. 1980,

Brenner et al. 1991, Holz et al. 1995, Meretoja et al. 1978a, Teixeira et al. 2004,

Watanabe et al. 1983, Watanabe et al. 1981). Data are recorded as the frequency of SCE

per cell and also may include the proportion of high-frequency cells (HFCs). Because

baseline levels of SCEs show considerable variation among individuals and between

studies, it is difficult to classify subjects into high, medium, or low categories (Albertini

et al. 2000).


Table 5-15. Sister chromatid exchange in lymphocytes from workers occupationally exposed to styrene Styrene exposure

mean (range) Results


Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)

SCE/cell (mean ± SD)

Exposure response Commentsc Meretoja et al. 1978a (Finland)

Polyester plastic manufacturing workers (laminators) (1−15 yr)


NR (≤ 300) NR (23–3,257) 5.3 ± 1.0 4.4 ± 0.6

Total population included 16 laminators and 6 controls, but results not available for all subjects Controls not matched, but had similar age range; all subjects were male No previous exposure to known clastogenic agents SCE were not correlated with smoking 15–25 metaphases/subject Statistics: Student’s t-test

Andersson et al. 1980 (Sweden)

Reinforced-plastics boat factory workers (0.3−12 yr)


Controls 21

(mg/m3 × yr) 575 (6–1,589)

1,204 (710–1,589 137 (6–283)

NR

8.4 ± 1.3* 8.7 ± 1.3* 8.2 ± 1.3

7.5 ± 1.1

Total population included 39 exposed and 41 controls, but results not available for all subjects Subjects interviewed about health history Controls matched on age and included 3 groups (office, assembly shop, and workshop) from the same factory; all subjects were male 25 metaphases/subject Statistics: Student’s t-test


Group 1: Reinforced-plastics boat factory (workshop 1) Group 2: Polyester resin

Group 1: exposed 9 controls 5

Group 2: exposed 7 controls 8

< 70 (1–211)

36 (NR)

(mg/L) 647 (90–4,300)

32 (5–115)d

526 (300–1,360)

32 (5–115)d

7.8 ± 1.6 7.6 ± 1.2

6.7 ± 0.8 7.6 ± 1.2

Controls matched on age and sex, all subjects in group 1 were male; Group 2 included males and females Exposure varied depending on the work in workshop 1 but was stable in workshop 2 17–50 metaphases/subject

9/29/08 347


348 9/29/08

Styrene exposure mean (range) Results


Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc board workers (workshop 2) (NR)

Mitomycin C treatment did not increase the number of SCE in exposed or controls Statistics: Student’s t-test or Chi-square


Fiber reinforced-plastics boat factory workers in 2 workshops (groups A & B) (1 mo–30 yr)

Exposed total 18 group A 10 group B 8

Controls 6

40–50 (NR)

(mg/L) 332 (0–1,041) 399 (0–1,041) 249 (8–999)

8.9 ± 1.4 8.6 ± 1.2 9.1 ± 1.8

8.5 ± 1.0 Exposure response total urinary metabolites: r = 0.525, P < 0.05

Controls matched on age; all subjects were male Subjects interviewed about occupational and medical history, and smoking habits; more smokers in exposed group than controls (72% vs. 50%) Workers not exposed to other industrial chemicals SCE significantly higher in exposed smokers than exposed non-smokers; no difference in controls 9–30 metaphases/subject Statistics: Mann-Whitney U test, t-test (two-tailed)

Camurri et al. 1983, 1984 (Italy)

Reinforced unsaturated polyester resin manufacturing workers in 9 plants (1–22 yr)

Plant 1 3 Control 3

Plant 2 4 Control 4

Plant 3 4 Control 6

Plant 4 5 Control 6

Plant 5 6 Control 6

NR [7–9]

NR [16–23]

NR [23–34.5]

NR [34.5–46]

NR [46–57.5]

(mg/L) NR (45–75)

NR (65–133)

NR (170–694)

NR (151–786)

NR (340–671)

12.7 ± 0.7 12.1 ± 1.3

12.7 ± 0.4* 11.7 ± 0.6

10.9 ± 1.0 9.7 ± 1.5

10.3 ± 0.9 9.7 ± 1.5

11.8 ± 0.5** 10.8 ± 0.3

Data described for 6 plants in 1983 publication; all data described in 1984 publication Controls matched for age, sex, and smoking Workers exposed to other industrial chemicals (e.g., organic peroxides, solvents, and dyes) 16–74 metaphases/subject SCE did not correlate with smoking habits Statistics may have been based on cell as unit rather than individuals Significant differences in SCE at concentrations ≥ 200 mg/m3 (46.9 ppm) with steep increases


9/29/08 349



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc Plant 6 2 Control 2

Plant 7 2 Control 2

Plant 8 4 Control 2

Plant 9 7 Control 4

NR [57.5–69]

NR [69–80.5]

NR [80.5–92]

NR (>92)

NR (615–777)

NR (489–828)

NR (504–909)

NR (389–1,108)

19.6 ± 5.2 10.3 ± 0.1

16.1 ± 1.6* 10.4 ± 0.0

16.1 ± 0.13** 9.7 ± 1.5

15.1 ± 0.5*** 8.5 ± 1.1

occurring at about 250 mg/m3 [57.5 ppm] Statistics: Student’s t-test

Hansteen et al. 1984 (Norway)

Glass-fiber reinforced polyester plant workers (2 groups based on exposure levels) (NR)


Controls 9

13.2 (2–44) 7.5 (2–13)

22.3 (14–44)

NR (200–1,200)e

6.6 6.9 6.0

6.5

Controls matched on age, sex, and smoking Low exposure SCE were not significantly higher in smokers vs. non-smokers (total, exposed, or control groups) Statistics: Fisher-Irwin test, Wilcoxon two-sample ranking test




[23 (8–60)]

(mM) 2.0 (0–7.3)

7.6 ± 0.2f 7.4 ± 0.2

Exposure response No correlation with exposure extent or duration

Controls matched on sex and smoking. No differences between controls and exposed in alcohol consumption, drug intake, vaccinations, recent viral infections, and previous occupational exposure to chemicals. SCE significantly higher in smoking controls than non-smoking controls Statistics: Student’s t-test, analysis for exposure response not reported

Kelsey et al. 1990

Reinforced-fiberglass plastic boat building

Smokers exposed 7

[48] (NR)

275 (NR)

7.2 ± 1.3

All subjects were male except 1 female in controls; did not differ from exposed workers with respect to age, smoking, coffee or alcohol consumption, or


350 9/29/08



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc (United States)

workers (smokers 8.6 yr, nonsmokers 7.2 yr)

controls 8


[2.3] (NR)

[53] (NR)

[0.76] (NR)

21 (NR)

323 (NR) 13 (NR)

7.2 ± 1.3

6.5 ± 0.7 6.2 ± 0.9 Exposure response No increase with styrene air levels or urinary metabolites

recent viral infections or vaccinations (none reported). Some exposure to styrene in the control group Styrene exposure did not affect SCE in high SCE frequency cells (HFC); however, smokers (total population) had significantly higher SCE in HFC than non-smokers. Statistics: Student’s t-test, ANOVA for exposure response analysis


Reinforced plastic workers (smokers – 6.4 yr, non-smokers – 7.2 yr)




[~70] (NR)g

(mM/L) 9.4 (< 1–21.5)

11.0 (< 1–16.6)

6.5 (< 1–21.5)

11.4 ± 1.7 12.4 ± 1.6

12.2 ± 1.7 12.9 ± 1.4

10.2 ± 0.8 11.4 ± 1.5

Controls from a research institute Age, sex, smoking, general health, alcohol consumption, drug intake, viral infections, vaccinations, and exposure to other chemicals considered. SCE higher in smokers than non-smokers (in both the exposed and control groups). Statistics: Student’s t-test (one-tailed)

Brenner et al. 1991 (United States)

Reinforced fiberglass plastic boat workers (2.7 yr)

Exposed total 10 high 4 low 6 Controls 9

11.2 (1–44) 27.2 (7–44) 6.8 (1–18)

243 (96–2,496) 523 (96–2,496) 176 (96–496)

9.7 ± 0.4f 10.0 ± 0.6 9.4 ± 0.5 10.1 ± 0.4

Controls were library workers at a university and differed by sex and current smoking (which were retained in the analysis), education, and medication (which could not be retained in the analysis due to small numbers of subjects). No differences with respect to age, caffeine and alcohol intake, recency of colds or X-rays, other tobacco-related exposures, and exposure to wood smoke or solvents.


9/29/08 351



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc 2 slide readers (25 metaphases each/subject) No effect on HFCs Statistics: Wilcoxon rank-sum test, Chi-square, ANOVA, which included gender, smoking, exposure and educational status, used to evaluate exposure response in 3 exposure groups


Reinforced-plastics industry workers from 32 workshops (NR)

Past exp. (index pts) Laminators low 12 high 13


Controls other factory 19 plastics factory 12

All subjects exposed 70 controls 31

43 (5–182)

11 (1–133)

(mM) 2.2 (NR)e

7.7 ± 1.2 7.4 ± 1.4

7.3 ± 1.0 7.6 ± 1.1

6.9 ± 0.8 7.2 ± 1.0

NR NR

Exposure response F = 4.66, P = 0.016 for male laminators who smoked compared to other male smoking workers. No overall association with

Total population consisted of 248 exposed workers, including 154 laminators and 63 controls (for cytogenetic analysis). SCE results available on subset past exposure index not available on all subjects 2 control groups: 1 from the plastics industry and 1 from other industries Past exposure estimated using a grading scale based on exposure duration, urinary metabolites, and air concentrations. Exposure groups divided into two subsets based on past exposure Age and smoking significantly associated with SCE in regression analysis Statistics: Regression analysis; no details provided


352 9/29/08



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc styrene exposure

Yager et al. 1993 (United States)

Boat manufacturing workers (6.4 yr)

Exposed total 46 high 11 medium 20 low 15

[15] (NR) [40] (NR) [10] (NR) [0.8] (NR

NR

6.4 ± 0.1f 6.9 ± 0.3 6.5 ± 0.2 6.1 ± 0.2 Exposure response Air: r = 0.4, P < 0.01 Breath: r = 0.5, P = 0.001

No controls; longitudinal study; subjects’ exposure determined from personal air monitors and concentrations in exhaled breath on 7 days over a 1-year period Smokers equally distributed over all groups SCEs analyzed twice (replicates) for most subjects SCEs significantly increased with smoking and exposure to styrene (smoking accounted for 62% and styrene 25% of the total variability) Statistics: linear regression analysis (including smoking, alcohol intake, some dietary factors, drug intake, immunizations, infections, exposures from hobbies and home repairs) and occupational history for exposure response

Hallier et al. 1994 (Germany)

Reinforced-plastics workers (8 yr)

Total laminators 14 laminators 9h formers 14 controls 20

Smokers laminators 4 formers 13 controls 10

Nonsmokers laminators 10

37 (29–42) 15 (12–21)

10 (NR)

(mg/L) 652 (100–1,610) 187 (100–400)

NR

10.1 ± 1.0* 8.2 ± 0.99 7.6 ± 1.5 6.6 ± 1.0

9.59 ± 0.77* 7.42 ± 1.27 7.23 ± 1.00

10.25± 1.08*

All subjects were male No occupational exposure to hazardous chemicals SCE significantly higher in smoking controls vs. nonsmoking controls SCE was lower in the 9 laminators retested ~1 year after styrene exposure was reduced by half, but controls were not retested Statistics: Mann-Whitney rank U test, Wilcoxon test for laminators after reducing exposure


9/29/08 353



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc controls 10

5.98 ± 0.06




Controls 23

[16 (0–138)] [20 (0–138)] [12 (0–34)]

NR

10.2 ± 0.9*** 10.1 ± 0.8*** 10.6 ± 0.6***

5.6 ± 0.3 Exposure response No correlation with exposure duration

Controls matched on age, sex, and smoking Workers divided into 2 groups with similar working conditions, but blood samples were taken 1 wk apart Subjects questioned about health status, exposure to X-rays, drug use, and smoking and alcohol habits; blood samples tested for some viral infections Workers also exposed to dichloromethane [genotoxin] Significant effect (P = 0.03) of smoking in controls but not the exposed HFCs (> 9 SCEs/cell) were > 14-fold higher in exposed workers than controls; no effect of smoking on HFCs Statistics: one-tailed Mann-Whitney U test

Van Hummelen et al. 1994 (Belgium)

Fiberglass-reinforced-plastics workers (2.9 yr)



[7 (0.5–25)]

102 (11–649)

5.47 ± 0.10f 5.62 ± 0.32

4.41 ± 0.20 4.94 ± 0.45

Exposure response No correlation with styrene air levels or

Study consisted of 52 exposed and 24 nonexposed workers, but cytogenetic results were not available on all subjects because of technical problems All subjects were male; control group selected from a factory that produced and repaired pallets Subjects were interviewed regarding exposure to potential carcinogens and mutagens, smoking habits, diet, viral infections, vaccinations, chemotherapy, and X-rays. Exposed were


354 9/29/08



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc urinary metabolite levels

significantly older (31 vs. 27), consumed less alcohol (1 vs 2.6 drinks/d), and had more recent X-rays (0.6 vs. 3.6 yr ago) Relatively low exposure Smoking significantly increased SCE frequency; no correlation of SCE with age, medical history, or other lifestyle factors No association between styrene exposure and HFC was observed. Statistics: two-tailed Mann-Whitney U test

Artuso et al. 1995 (Italy)

Fiber-reinforced-plastics boat building workers (NR)

Lab 1 low 13 high 19 controls 21

Lab 2 low 9 high 4 controls 13

NR [0.5–28] NR [20–320]

NR [0.5–28] NR [20–320]

NR

2.82 ± 0.12*f 3.01 ± 0.12* 2.38 ± 0.10

6.47 ± 0.50 7.32 ± 0.81 5.44 ± 0.26

Exposure response Tests for linear trend significant for both labs Multiple regression (RR, 95% CI): Low: 1.22 (1.05–1.43)

All subjects were males; controls matched for age and smoking and from the same area as exposed Subjects questioned about working activity, recent illness, exposure to X-rays, use of drugs, alcohol, coffee and smoking habits. X-rays were more frequent among controls 3 scorers from 2 labs used No significant association with smoking, alcohol consumption and diagnostic X-rays Statistics: t-test with Tukey adjustment, multiple linear regression, which included exposure, smoking, alcohol drinking, and exposure to diagnostic X-rays, with adjustment for age and slide reader


9/29/08 355



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc High: 1.26 (1.07–1.47)

Holz et al. 1995 (Germany)

Styrene production plant workers (1–34 yr)

Total Exposed 25 Controls 25

Smokers Exposed 17 controls 13


NR (0.01–> 0.9)

NR [≤ 0.01]

13.3–43.9 (NR)i

4.3–5.5 (NR)

10–49.4 (NR) 5.6–6.3 (NR)

20.3–32.4 (NR) 2.8–4.6 (NR)

9.27 ± 1.24 9.24 ± 1.24

9.38 ± 1.37 9.67 ± 1.36

9.04 ± 0.99 8.87 ± 0.96

Controls from the same plant matched for age and sex and had similar smoking habits Controls were exposed to low levels of styrene Subjects questioned about alcohol consumption, smoking, drug use, and exposure to aromatic hydrocarbons outside the workplace Workers exposed to aromatic hydrocarbons: ethylbenzene (highest exposure), benzene, toluene, and xylene 15 metaphases/subject Statistics: Student’s t-test

Rappaport et al. 1996 (United States)

Reinforced plastic boat manufacturing workers (≥ 1 yr)

Exposed smokers 22 nonsmokers 24

[17 (0.4–51)] [12 (0.2–54)]

NR

6.73 ± 0.22f 6.07 ± 0.140 Exposure response Styrene: r = 0.39, P < 0.1 SO– all: r = 0.23, NS SO– smokers: r = 0.81, P = 0.015

No controls; gender ratios not provided Correlation analysis suggested that styrene-7,8-oxide (SO) was 2,000 times more effective than styrene in producing SO biomarkers (albumin or DNA adducts) Statistics: ANOVA, Pearson’s correlation, multiple linear regression

Karakaya et al. 1997 (Turkey)

Furniture workers (10 yr)


30 (20–300)

207 (14–1,482)e

12 (0–38)e

6.2 ± 1.6** 5.23 ± 1.23

Total population consisted of 53 exposed subjects, but SCE results were not available for 9 subjects due to poor sample preparations.


356 9/29/08



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc Smokers exposed 29 controls 29


6.8 ± 1.3 5.88 ± 0.76

5.2 ± 1.4** 3.66 ± 0.52 Exposure response No significant correlation with exposure duration (although a trend was noted) for UT levels

All subjects were male; controls were university office workers matched on age and smoking Subjects interviewed about occupational, family, and dietary history. No information on other workplace exposures SCE significantly higher (P < 0.01) in smokers vs. nonsmokers in controls and exposed, but not affected by age Urinary thioethers (UT) were significantly higher in exposed than controls Statistics: ANOVA, Mann-Whitney U test

Biró et al. 2002 (Hungary)

Oil refinery workers (NR)


NR NR 7.9 ± 0.3* f 6.4 ± 0.2

Subjects interviewed about age, medication, smoking and drinking habits, and medical and work histories More smokers in exposed (80%) vs. controls (20%) SCE was higher in smokers but no separate analysis was conducted for smokers and nonsmokers Statistics: Student’s t-test

Laffon et al. 2002a (Spain)

Fiberglass-reinforced plastic production workers


< 20 (NR)

313–353 (NR) 3.5 ± 0.06** f

2.6 ± 0.05 Exposure response

All subjects were male; controls were university employees, and workers were employed at least 7 yr


9/29/08 357



Study population

(yrs employed)a

Number of subjects

Air (ppm)b


(mg/g creatinine)


Exposure response Commentsc (17 yr) Positive correlation

with exposure duration (P ≤ 0.01)

Subjects queried about smoking and alcohol habits, medication, recent infections and vaccinations, diagnostic tests such as X-rays, and previous occupational exposure to chemicals SCE significantly increased with number of cigarettes/d in exposed but not controls More controls smoked (53%) than workers (36%), but workers had smoked longer Workers also exposed to organic peroxides, acetone, and dichloromethane SCE higher in smokers in exposed group HFC significantly increased in exposed group Statistics: Student’s t-test, one-way ANOVA

Teixeira et al. 2004 (NR)

Reinforced-plastics workers from 2 small plants (1–30) yr


Men exposed 18 controls 18

Women exposed 10 controls 10

27 (2–91)

401 (47–1,490)g

7.18 ± 0.34* f 6.30 ± 0.25

7.25 ± 0.51 5.99 ± 0.31

7.04 ± 0.28 6.86 ± 0.036

Controls were office workers who were similar in age, sex ratio, and smoking habits as the exposed groups Subjects queried about smoking and alcohol habits, medication, diet, X-ray exposure, and previous occupational exposure to chemicals Exposed group also exposed to low levels of toluene and acetone (< 1% styrene concentrations) ≥ 25 metaphase/individual Statistics: t-test

* P < 0.05, ** P < 0.01, *** P < 0.001. HFC = high SCE frequency cells, NR = not reported, SCE = sister chromatid exchange. a Study population includes both sexes unless otherwise noted.


358 9/29/08

b [Bracketed data were converted from mg/m3 to ppm (1 mg/m3 styrene ≈ 0.23 ppm).] c Control group included unexposed workers from the same plant as the exposed except where noted otherwise. Potential confounders (e.g., exposures to other chemicals, recent infections, vaccinations, etc.) are noted as identified by the study authors. d Values reported for 5 controls but the group(s) were not identified. e Sum of mandelic and phenylglyoxylic acids. f Mean ± SE. g Air concentration was estimated from urine mandelic acid levels. h Subset of exposed workers that were sampled one year after reducing exposure. i Values represent the mean concentrations measured before and after the work shift.


9/29/08 359

The general limitations [i.e., small numbers of subjects and failure to control for potential

confounding factors] noted for chromosomal aberration and micronucleus studies also

apply to SCE studies. Most of the studies included control groups matched on one or

more of the following variables: age, gender, or smoking. Some studies that did not use

matched subjects controlled for variables (such as age, gender, and smoking habits) in the

analysis or reported that age, smoking, and gender distribution were similar between

groups. Only two studies (Sorsa et al. 1991) [it was not clear whether smoking and age

were controlled for in the dose-response regression analysis], and Biró et al. (2002) did

not meet those criteria. Biro et al. did report that ages were similar, but smoking habits

and gender differed between the exposed and referent groups. Ten studies reported that

smoking was significantly correlated with SCE (Hallier et al. 1994, Karakaya et al. 1997,

Kelsey et al. 1990, Laffon et al. 2002a, Maki-Paakkanen et al. 1991, Sorsa et al. 1991,

Tates et al. 1994, Van Hummelen et al. 1994, Watanabe et al. 1983, Yager et al. 1993),

while Meretoja et al. (1978a), Camurri et al. (1983, 1984), Hansteen et al. (1984),

Brenner et al. (1991), Artuso et al. (1995), Holz et al. (1995), Rappaport et al. (1996),

and Teixeira et al. (2004) did not find an association with smoking. Age was associated

with SCE in the study by Sorsa et al. (1991) but not in another study (Van Hummelen et

al. 1994). None of the studies reported that gender was a significant factor.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Two studies (Rappaport et al. 1996, Yager et al. 1993) had longitudinal study designs

(without controls), and compared styrene exposure measured at several times during a

one-year period with SCE frequency. Both studies reported positive correlations with

styrene exposure (levels in exhaled air and/or other biomarkers of styrene exposure).

Nine of the remaining 20 studies reviewed reported a significant increase in SCEs in

workers exposed to styrene compared with controls (Table 5-15). These nine studies

included Andersson et al. (1980), Camurri et al. (1983, 1984) [considered as one study],

Hallier et al. (1994), Tates et al. (1994), Artuso et al. (1995), Karakaya et al. (1997), Biro

et al. (2002), Laffon et al. (2002a), and Teixeira et al. (2004). In addition to the exposure-

response relationships observed in the longitudinal studies, Artuso et al. (1995) (exposure

level), Laffon et al. (2002a) (exposure duration), and Watanabe et al. (1983) (total

urinary metabolites) also reported significant exposure-response associations. However,


360 9/29/08

no associations were reported in other studies (Kelsey et al. 1990, Sorsa et al. 1991, Tates

et al. 1994 [for exposure duration], Van Hummelen et al. 1994, and Karakaya et al.

1997). Workers in the study by Tates et al. (1994) were exposed to dichloromethane;

however, no correlation was found between dichloromethane exposure and SCE levels.

Workers in other studies (Camurri et al. 1983, 1984, and Laffon et al. 2002a) were

exposed to other chemicals such as organic peroxides, dyes, and acetone. Andersson et

al. (1980) did not report results for all subjects and reported exposure as the product of

styrene concentration and years exposed. Hallier et al. (1994) found that SCE levels in

laminators decreased after technical and hygienic improvements reduced styrene levels

from 37 to 15 ppm. Biró et al. (2002) did not provide styrene concentrations or levels of

styrene metabolites in the urine.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

22

23

24

25

26

27

28

29

SCE levels were not significantly higher in exposed workers than controls in the studies

by Meretoja et al. (1978a), Watanabe et al. (1983, 1981), Hansteen et al. (1984), Mäki-

Paakkanen et al. (1991), Mäki-Paakkanen (1987), Kelsey et al. (1990), Brenner et al.

(1991), Sorsa et al. (1991), Van Hummelen et al. (1994), and Holz et al. (1995). There

were no clear differences in the number of subjects or mean styrene concentrations

between the positive and negative studies. However, most of the studies published prior

to 1994 were negative while most of the studies published after 1994 were positive. The

meta-analysis by Bonassi et al. (1996) was inconclusive regarding styrene exposure and

SCE.

5.4.5 Genetic polymorphisms and susceptibility to styrene-mediated genotoxicity 21 Individuals may vary in their susceptibility to styrene’s genotoxic effects because of

differences in the ability to activate and inactivate styrene or differences in DNA-repair

capacity. CYP2E1 is one of the primary enzymes involved in the metabolism of styrene

to styrene-7,8-oxide, and detoxification is mediated by mEH [also known as EPHX1] and

glutathione S-transferases (GSTs); conjugation of styrene with glutathione as a minor

detoxification pathway (see Section 5.1.3 for a detailed description of the metabolism of

styrene). Studies have been conducted in vitro with human lymphocytes exposed to

styrene and in styrene-exposed workers to evaluate whether polymorphisms in xenobiotic


9/29/08 361

metabolizing enzymes, DNA repair, or other critical pathways modulate genotoxic

damage.

1

2

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

5.4.5.1 In vitro studies 3 The findings of the in vitro studies are summarized in Table 5-16. Human lymphocytes

with polymorphisms in metabolic enzymes (coded for by CYP1A1, CYP2E1, GSTM1,

GSTT1, GSTP1, or mEH) or DNA-repair enzymes (coded for by hOGG1, XRCC1, or

XRCC3) were exposed to either styrene (usually at concentrations between 5,000 and

10,000 μM, although one study used 500 to 1,500 μM) or styrene-7,8-oxide (usually at

10 to 300 μM, although one study used 600 to 2,500 μM). Genotoxicity (single-strand

breaks, HPRT mutations, micronuclei, or SCE) was measured and compared among the

genotypes. [Most studies used only a small number of cells per genotype group. It is

difficult to draw any conclusions about the effects of specific polymorphisms in

modifying specific damage, because some of the polymorphisms were evaluated in only

one study, or conflicting results were observed when the polymorphism was evaluated in

several studies (e.g., GSTM1 and GSTT1). Interpretation of the GSTM1 and GSTT1

studies also is complicated because the studies looked at different end points (e.g., SCE,

HPRT mutations) or used different exposure agents (styrene or styrene-7,8-oxide), and

some studies looked at combinations of GSTM1 and GSTT1 genotypes.] Three studies

reported higher levels of genetic damage in a GSTP1 variant; [however, the studies varied

in the exposure (styrene or styrene-7,8-oxide), the end point affected (single-strand

breaks or micronuclei), and the variant in which the effect was observed].


Table 5-16. Genotype analyses in in vitro studies with styrene and styrene-7,8-oxide Exposure (µM)

Genotypes Polymorphisms

Number of donors Styrene

Styrene-7,8-oxide End points

Results (compared with + or wild type) Reference

CYP1A1 Msp I (m1, m2, m4)a

var. 4–10 wt 20–26

5,000–10,000 – SB more SSB in CYP1A1 m1 and m2 heterozygotes; less damage in m4 heterozygotes

Laffon et al. 2003a

CYP2E1 RsaI and DraIb

var. 1–6 wt 24–29

5,000–10,000 – SB more SSB in CYP2E1 DraI heterozygotes Laffon et al. 2003a

6–8 – 10–100 SB no genotype effect Buschini et al. 2003

5–16 5,000–10,000 – SB no genotype effect Laffon et al. 2003a

6–18 – 50–200 MN, SB more MN and SSB in cells with lower mEH activity

Laffon et al. 2003b

EPHX1 low, medium, high meH activity

4–9 – 100–300 MN, SB more MN in cells with higher mEH activity at 200 μM


6+/6– – 50–150 SCE no genotype effect Uüskula et al. 1995

3+/2– cell linesc – 600–2,500 HPRT mutation

more mutations in GSTM1-deficient cell lines

Shield and Sanderson 2004, 2001)

5+/9– – 10–100 SB no genotype effect Buschini et al. 2003

15+/12– 5,000–10,000 – SB no genotype effect Laffon et al. 2003a

17+/13– – 50–200 MN, SB no genotype effect Laffon et al. 2003b

GSTM1 null

8+/12– – 100–300 MN, SB no genotype effect Godderis et al. 2006

5+/5– – 50–150 SCE more SCE in GSTT1 null Ollikainen et al. 1998

21+/6– 5,000–10,000 – SB no genotype effect Laffon et al. 2003a

24+/6– – 50–200 MN, SB no genotype effect Laffon et al. 2003b

11+/3– – 10–100 SB no genotype effect Buschini et al. 2003

GSTT1 null

14+/6– – 100–300 MN, SB more MN with GSTT1 (considered a spurious effect)


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Exposure (µM) Genotypes Polymorphisms

Number of donors Styrene

Styrene-7,8-oxide End points

Results (compared with + or wild type) Reference

GSTM1 and GSTT1

combinations 5–7 500–1,500 – SCE more SCE in GSTM1 null/GSTT1 null at

high styrene level Bernardini et al. 2002

7 wt/7 var. – 10–100 SB more SSB in GSTP1 variants Buschini et al. 2003

4–18 5,000–10,000 – SB more SSB in GSTP1 105 variant Laffon et al. 2003a

4–18 – 50–200 MN, SB more MN (nonsignificant) in GSTP1 105 and 114 variant (combination)

Laffon et al. 2003b

GSTP1 codon 105 alone or in combination with codon 114d

3–10 – 100–300 MN, SB no genotype effect Godderis et al. 2006

hOGG1 codon 326e

7–13 – 100–300 MN, SB no genotype effect

XRCC1 codons 194f, 280e, 399e


XRCC3 codon 241e



+ = positive for gene; – = negative for gene; HPRT = hypoxanthine phosphoribosyltransferase; MN = micronuclei; SB = strand breaks; SCE = sister chromatid exchange; var. = variant; wt = wild type. a Predicted influence on the enzyme is increased inducibility (m1), increased activity (m2), and decreased activity (m4). b Predicted influence on the enzyme is unknown. c These studies used established human B lymphoblastoid cell lines instead of whole-blood lymphocyte cultures from donors. d Compared to the wild-type enzyme, the variant proteins show either a reduced half-life or a different catalytic efficiency toward organic substrates. e Low activity. f High activity.


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1 5.4.5.2 In vivo studies

The findings for studies that evaluated polymorphisms in metabolizing enzymes and

genotoxic effects in styrene-exposed workers are summarized in Table 5-17. Results

evaluating the relationship between polymorphisms and genetic damage in both non-

exposed and styrene-exposed workers are not discussed in this review. [Most of these

studies determined the genotypes of fewer than 50 styrene-exposed workers for several

polymorphisms, measured various genotoxic end points, and compared the amount of

genetic damage among genotype groups. As with the in vitro studies, it is difficult to

draw any conclusions regarding specific polymorphisms, because the findings were

conflicting, and the studies had many limitations. The number of individuals per

genotype was very small, limiting the statistical power to detect an effect. Many studies

did not adjust for potential confounders and made multiple comparisons, thus increasing

the possibility of obtaining false-positive results. ]

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One study of styrene-exposed workers evaluated polymorphisms in DNA-repair genes

and genotoxicity; however, this study included both styrene-exposed workers and

referents, and most of the analysis was of the whole population rather than specifically

the styrene-exposed populations. Kuricova et al. (2005) measured DNA adducts, single-

strand breaks, HPRT mutations, and chromosomal aberrations among 48 workers (16

males and 32 females) at a styrene-reinforced-plastics plant (the same population studied

by Vodicka et al. (2001a); see Table 5-17). Levels of damage were compared among

polymorphisms for XPD, XPG, XPC, XRCC1, XRCC3, and cyclin D1. Most of the

results were presented for the entire population (which also included 24 controls), but the

authors stated that styrene-exposed individuals with the XRCC1 399 wild-type genotype

had a lower frequency of chromosomal aberrations than individuals with the variant

genotype. [This was the only significant finding among the exposed population.]

[In addition to the limitations discussed (small numbers of subjects or cell lines, multiple

comparisons, potential confounding, conflicting results, and very few studies evaluating

similar end points and genotypes), the genetic susceptibility studies discussed evaluated

only single polymorphisms. The evaluation of multiple loci or several polymorphisms in


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the same pathway may be more informative for identifying populations sensitive to

styrene-mediated genotoxicity.]

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Table 5-17. Genotype analyses in vivo in workers occupationally exposed to styrene in association with biomarkers of genotoxicity


No. of exposed workers End pointsa Results for exposed workersb Reference

CYP1A1 MspI

44 (19 HPRT 29 SSB)

SSB, CA, HPRT mutation

no effects reported Vodicka et al. 2001a

44 (19 HPRT 29 SSB)


more HPRT mutations in DraI heterozygotes; more SSB in both RsaI and DraI heterozygotes; no effects for CA.


19 DNA adducts, HPRT

more adducts in heterozygotes, no effect on adducts

Vodicka et al. 2003

CYP2E1 RsaI and DraI

28 SCE, MN no effects reported Teixeira et al. 2004

44 (19 HPRT 29 SSB)



48 SSB no effects reported Buschini et al. 2003

19 DNA adducts, HPRT mutation

no effects reported Vodicka et al. 2003

EPHX1 low, medium, high mEH activity

28 SCE, MN decreased SCE for medium mEH activity; no effect on MN

Teixeira et al. 2004

44 (19 HPRT 29 SSB)



14 SSB, SCE, MN no effects reported, but increased PRIc was observed in GTSM1 null

Laffon et al. 2002a

48 SSB fewer SSB in GSTM1 null Buschini et al. 2003




GSTM1 null

95 MN, CA no effects reported Migliore et al. 2006b

44 (19 HPRT 29 SSB)



14 SSB, SCE, MN no effects reported Laffon et al. 2002a

48 SSB more SSB in GSTT1 null Buschini et al. 2003



GSTT1 null



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No. of exposed workers End pointsa Results for exposed workersb Reference

95 MN, CA significantly higher frequency of MN and non-significant increase in CA

Migliore et al. 2006b

44 (19 HPRT 29 SSB)

SSB, CA, HPRT marginal effect on HPRT mutationd no effects reported for SSB and CA



more HPRT mutations in heterozygotes, no effect on adducts

Vodicka et al. 2003


GSTP1 codon 105 alone or in combination with codon114

95 MN, CA no effects reported Migliore et al. 2006b aCA = chromosomal aberrations, MN = micronuclei, SSB = single-strand breaks. bResults reported for exposed populations only. Some studies did report association between polymorphisms and genetic damage in the total population (controls and exposed workers) or in controls only. cPRI = proliferation rate index = (MI + 2MII + 3MIII)/N, where MI, MII, and MIII = the number of metaphases in first, second, and third or subsequent divisions, and N = the total number of metaphase scored in the SCE assay. dThe effect was observed only when outliers were included.

1 5.4.6 Summary of styrene and styrene-7,8-oxide genotoxicity Results from in vitro studies and in vivo studies in experimental animals and humans are

summarized in Table 5-18. DNA adducts (primarily O6-deoxyguanosine and N7-

deoxyguanosine) have been detected in the liver and lungs of rats and mice exposed to

styrene by inhalation or i.p. injection. O6-deoxyguanosine, N2-guanine, and N1-adenine

adducts have been detected in lymphocytes of workers occupationally exposed to styrene.

In vitro and in vivo studies indicated that styrene could induce DNA damage including

single-strand breaks. Mutation studies in bacteria were mostly negative without metabolic

activation, but some studies were positive with metabolic activation. In vitro mutation

studies with eukaryotic cells gave mixed results. No mutation studies of styrene-exposed

experimental animals were reviewed. A few studies investigated HPRT- and GPA-locus

mutations in styrene-exposed workers and reported inconclusive to weak positive results.

One study was positive for HPRT mutations but these workers also were exposed to

dichloromethane. In vitro studies indicate that styrene can cause chromosomal

aberrations, SCE, and micronuclei; whereas, in vivo studies in rodents were positive for

SCE only. A meta-analysis of studies of occupational exposed workers reported a

positive association between styrene exposure level (higher levels) and chromosomal

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aberration frequency. Studies in occupationally exposed workers show conflicting

responses with SCE and micronuclei formation.

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Table 5-18. Genetic and related effects of styrene In vivo

Effect In vitro Rodents Humans DNA adducts NSRa + + DNA damageb +1 + +

(+) – NA ± NA

Mutations bacteria lower eukaryotes mammalian cells ±

NS NA

Chromosomal aberrations + – (+) Sister chromatid exchange (+) + ± Micronuclei + – ± Aneuploidy or polyploidy ± +1 – + = predominantly positive results; +1 = positive results in the only study reviewed; ± = similar number of positive and negative results or multiple studies with positive and negative results; (+) = weakly positive results; – = predominantly negative results. NA = not applicable; NSR = no studies reviewed. a Studies with styrene-7,8-oxide did cause DNA adducts in vitro. b Includes alkali-labile sites and single-strand breaks.


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5.5 Mechanistic studies and considerations 1 Several recent publications reviewed the possible mechanisms of styrene-induced

carcinogenicity. Both genotoxic and epigenetic processes have been considered. IARC

(2002) proposed two likely mechanisms for styrene carcinogenicity: (1) DNA damage in

target tissues resulting from metabolic conversion of styrene to styrene-7,8-oxide and (2)

the cytotoxic effects of styrene in the lungs of mice. IARC did not consider these

mechanisms to be mutually exclusive and suggested that the interspecies differences in

the metabolism of styrene and styrene-7,8-oxide in rats and mice were likely important.

In addition, Cruzan et al. (2002) and The Harvard Center for Risk Analysis (Cohen et al.

2002) concluded that cytotoxicity and subsequent hyperplasia of lung cells must play a

key role underlying development of lung tumors in mice, and they proposed several

potential mechanisms that could explain how styrene exposure could cause development

of hyperplasia in the mouse lung but not the rat lung. Cohen et al. (2002) proposed that

the species differences between mice and rats (assuming that the lung tumors are caused

by styrene-7,8-oxide) could be due to a combination of higher rates of styrene-7,8-oxide

accumulation and greater susceptibility of the mouse lung to epoxides. IARC (2002)

noted that mice are considered to be more susceptible to induction of lung tumors by

epoxides and chemicals capable of being metabolized to epoxides than are rats, based on

findings of lung tumors in mice but not in rats when both species were exposed to

ethylene oxide, 1,3-butadiene, isoprene, or chloroprene. Cruzan et al. (2002) proposed

that interspecies differences in styrene toxicity are most likely explained through CYP2F-

generated metabolites such as 4-vinylphenol.

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Some (but not all) studies in experimental animals reported increased incidences of

tumors of the mammary gland or lymphatic system in rats (see Section 4), and increases

in mortality or incidence of pancreatic cancer and lymphohematopoietic malignancies

have been reported in some studies of styrene-exposed workers (see Section 3). However,

no styrene-specific mechanistic studies or reviews of the mammary gland, pancreas, or

lymphohematopoietic system as possible tumor sites were identified. (See Section 5.2 for

a discussion of prolactin, styrene exposure, and breast cancer). The following sections

discuss mechanistic considerations related to genotoxicity (Section 5.5.1), gene

expression and apoptosis (Section 5.5.2) , oxidative stress (Section 5.5.3), cytotoxic


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effects of styrene on mouse lung (Section 5.5.4), and selected styrene analogues (Section

5.5.5).

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Styrene-7,8-oxide, a primary and genotoxic metabolite of styrene, is listed in the Report

on Carcinogens as reasonably anticipated to be a human carcinogen based on sufficient

evidence in experimental animals (NTP 2004), and IARC (1994b) concluded that there

was sufficient evidence in experimental animals for its carcinogenicity. Styrene-7,8-oxide

and other epoxides, or epoxide-forming chemicals, are reactive compounds. Epoxides

have been associated with lung, liver, harderian gland, and circulatory system neoplasms

in mice; Zymbal’s gland and brain tumors in rats; and mammary gland and forestomach

tumors in rats and mice (Melnick 2002). Dunnick et al. (1995) and Bennett and Davis

(2002) reviewed findings from NTP’s carcinogenesis studies and reported that epoxides

or chemicals metabolized to epoxides were associated with mammary tumors in rodents.

Bennett and Davis hypothesized that the mammary gland may be efficient in

metabolizing chemicals to their epoxides. However, these authors also noted that not all

epoxides or epoxide-forming chemicals were associated with mammary tumors. Styrene-

7,8-oxide administered by oral gavage induced high incidences of both benign and

malignant tumors of the forestomach in both sexes of rats (three strains tested) and in one

strain of mice (IARC 1994b). One of the rat studies also included prenatal exposure

followed by postnatal gastric lavage. Lijinsky (1986) also reported liver tumors in male

mice in the low-dose group only. Lung and mammary tumors were not increased in these

studies. No inhalation carcinogenicity studies have been conducted with styrene-7,8-

oxide. However, styrene-7,8-oxide has been measured in the blood of rats and mice

following oral and i.p. administration (IARC 1994b). No reports of the levels of styrene-

7,8-oxide in the lungs of rats or mice exposed to styrene were identified, but a PBPK

model indicated that oral administration of styrene-7,8-oxide at 275 mg/kg per day would

result in higher lung levels of styrene-7,8-oxide than from metabolism of styrene

administered at 40 ppm by inhalation (Sarangapani et al. 2002) (see Section 5.3.4 for the

metabolic assumptions for this model).

An increased incidence of lung, liver, mammary gland, and lymphatic neoplasias has

been reported for some studies in experimental animals (see Section 4), although the


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results were not consistent across studies. Increases in mortality or incidence of

lymphohematopoietic malignancies and tumors at some other sites (such as the pancreas)

have also been reported in some studies of styrene-exposed workers (see Section 3).

However, no styrene-specific mechanistic studies or reviews of the mammary gland,

pancreas, or lymphohematopoietic system as possible tumor sites were identified. (See

Section 5.2 for a discussion of prolactin, styrene exposure, and breast cancer).The

following section discusses mechanistic considerations related to genotoxicity, gene

expression and apoptosis, oxidative stress, cytotoxicity in mouse lung, and studies of

selected styrene analogues.

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5.5.1 Genotoxicity 10

Some DNA adducts are highly xenobiotic-specific DNA lesions that can alter DNA

ultrastructure. IARC (2002) noted that a potential mechanism for the carcinogenicity of

styrene is based on covalent binding of the DNA-reactive metabolite styrene-7,8-oxide.

DNA adducts formed with styrene-7,8-oxide include N7-guanine, N3-adenine, O6-

guanine, N2-guanine, N1-adenine, N6-adenine, and N3-cytosine (see Section 5.4.1).

Adducts associated with oxidative damage (e.g., 8-hydroxy-2'-deoxyguanosine) also have

been reported in styrene-exposed workers (Marczynski et al. 1997a). N7-guanine adducts

are the predominant type, but are repaired in vivo, whereas O6-guanine adducts occur at a

much lower frequency but are more persistent (see Section 5.4.2.1). N7-guanine and N3-

adenine adducts may result in depurination or may cause single-strand breaks. Because

DNA polymerase preferentially adds an adenine opposite an apurinic site, N7-guanine

adducts may result in G·C→A·T transitions, and N3-adenine adducts may result in

A·T→T·A transversions (Loeb and Preston 1986). The other adducts occur at base-

pairing sites and may cause the following specific base-pair mutations: (1) O6-guanine,

G·C→A·T transition, (2) N2-guanine, G·C→A·T transversion (via incorporation of

deoxythymidine triphosphate opposite the adduct) (Zang et al. 2005b), (3) N1-adenine,

mutations at A·T base pairs (via blockage of a central hydrogen bonding site of the

adenine residue), (4) N3-uracil, G·C→A·T transition and, to a minor extent, G·C→T·A

transversion (via conversion to the N3-cytosine adduct) (Zhang et al. 1995), and (5) N6-

guanine, A·T→G·C transition. A·T→G·C transition was the dominant type of mutation in


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both styrene-7,8-oxide–exposed HPRT mutant clones (Bastlová and Podlutsky 1996) and

in a site-specific mutation study in which a styrene-7,8-oxide adduct at the N6-position of

adenine was inserted at codon 61 in the N-ras gene (Latham et al. 1993). Weak

mutagenicity was observed when S-styrene-7,8-oxide was bound at the α-carbon of

styrene-7,8-oxide to the adenine in the second position of the codon, while the R-

enantiomer bound at that position blocked replication of the single-stranded DNA

template almost completely, and no mutagenicity was found when either the R- or the S-

enantiomer was bound to the adenine in the third position of codon 61. The β-N6-dA

styrene-7,8-oxide adducts have been examined as to site-specific mutagenesis in E. coli.

These data indicate that the β-N6-dA adducts do not have significant deleterious effects

on replication competence (Kanuri et al. 2001).

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The actions of native and various site-specific mutants of HIV-1 reverse transcriptase

have been examined in vitro on DNA templates modified with α-N6-dA adducts. For the

native enzyme, activity is dependent on both the chirality of the N6-dA adducts and their

sequence contexts. Replication is possible but is terminated 3 to 5 nucleotides after

translesion synthesis and before reaching the end of the template (Latham and Lloyd

1994). Eight mutants of reverse transcriptase also terminate synthesis on these styrene-

7,8-oxide–adducted templates. The sites of termination occur primarily 1 and 3 bases

following adduct bypass, when the lesion is positioned in the major groove of the

template-primer stem (Latham et al. 2000).

Similar replication assays have been performed using E. coli Klenow fragment,

Sequenase 2.0, T4 polymerase holoenzyme, polymerase α, and polymerase β, in vitro. In

all instances, lesion bypass is sensitive to both the local sequence context and the

chirality of the α-N6–dA styrene-7,8-oxide adducts. For example, in the 5'-AXG-3'

sequence, adducts having R-stereochemistry are bypassed, whereas stereochemically-

identical lesions in other sequence contexts are often poor substrates. Similarly, R- vs. S-

α-N6–dA adducts introduced within identical sequences are often bypassed

nonequivalently. The degree of adduct-directed termination and translesion synthesis

during replication is also dependent on the choice of polymerase. Templates that are poor

substrates for bypass synthesis with one enzyme often read through much more


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efficiently when a different polymerase is used (Latham et al. 1995). Similar studies have

been conducted using reconstituted E. coli Pol III. Replication is poorly processive and

strongly terminated by styrene-7,8-oxide lesions in 33-mer templates, although the same

enzyme showed efficient bypass of the same adducts in M13 DNA (Latham et al. 1996).

No data are available regarding replication by Y-family polymerases, in vitro.

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Single-strand breaks were observed in most studies of styrene-exposed workers and

occurred in a concentration-dependent manner (see Section 5.4.4.2). Most studies have

reported an increase in chromosomal aberrations in styrene-exposed workers, and

exposure-response relationships have been observed in several studies. A meta-analysis

(Bonassi et al. (1996) of studies published prior to 1996 found a positive association

between styrene exposure (greater than 30 ppm for an 8-hour time-weighted average) and

chromosomal aberrations. The data on mutations and other types of genetic damage in

humans are conflicting. Most recent studies have reported higher levels of sister

chromatid exchange in styrene-exposed workers than in controls, but the study

populations were small, and potential confounding was not always addressed.

As described in Section 4, styrene exposure caused lung tumors in mice but not in rats. In

vivo experiments in rodents have shown that styrene exposure can cause DNA adducts in

lung and liver in mice and rats (see Section 5.4.3.1 and Table 5-7 for a description of

these studies). Comparison between animal studies is difficult because different species,

organs, methods of detection, routes of administration, and exposure levels were used.

Moreover, most genotoxicity studies in animals are short-term, and humans are exposed

for long time periods. No correlation of adducts with tumor incidence has been observed

(Nestmann et al. 2005), suggesting that other mechanisms of carcinogenicity may also be

important. Boogaard et al. (2000b) reported that styrene had a low covalent binding index

(CBI) relative to other known genotoxicants; the hepatic CBI (at 42 hours) was 0.19 in

rats and 0.44 in mice, and the pulmonary CBI (pooled 0 and 42 hours) was 0.17 in rats

and 0.24 in mice. The low levels of styrene-7,8-oxide adducts in the forestomach as the

target tissue for styrene-7,8-oxide in rats and mice were judged to be insufficient to

account for its carcinogenic activity by a strictly genotoxic mechanism (Phillips and

Farmer 1994) (see Section 5.4.3.1). However, the reported levels of DNA binding varied


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by factors of 20 to 50 among the studies. The reasons for the discrepancies were not

completely understood, according to the authors; however, some of the variability could

be attributed to differences in administration routes, measurement methods, and losses

through depurination.

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Koskinen et al. (2001a) compared the formation of β-N1-adenine adducts resulting from

styrene exposure in mice and humans. They reported that exposure of mice to styrene at

750 mg/m3 [173 ppm] resulted in formation of 1 β-N1-adenine adduct per 109 normal

nucleotides, while exposure of humans at 76 mg/m3 [17.5 ppm] resulted in 0.8 adducts

per 109 nucleotides [the results are reported in Table 5-10 as 0.08 adducts per 108

nucleotides].

5.5.2 Gene expression and apoptosis 11 The effect of styrene-7,8-oxide on the expression of genes involved in the cell cycle and

in regulation of apoptosis was studied in white blood cells exposed to styrene-7,8-oxide

at a concentration of 50 or 200 μM (Laffon et al. 2001a). mRNA and reverse

transcription polymerase chain reaction were used to analyze the expression of the genes

involved in cell-cycle arrest in response to DNA damage (p53, p21) or in control of

apoptosis (bcl-2 and bax). Apoptotic events were detected by the DNA fragmentation

assay. Data for expression were presented only in the form of graphs for individual

donors (2 men and 2 women described as healthy nonsmokers aged 23 to 30). The

authors reported high interindividual variation in the expression of studied genes, with no

consistent pattern of increased or decreased expression. The authors did describe a

difference in the cytokinesis block proliferation index (CBPI). All CBPI values for

control cultures and low-exposure cultures were between 1.94 and 2.04, while the values

for high-exposure cultures ranged from 1.67 to 1.78 and were significantly lower than in

the controls (P < 0.01), indicating a delay in cell-cycle kinetics. The authors suggested

that exposure to high levels of styrene-7,8-oxide might induce a delay in the cell cycle,

which could allow the DNA repair system to act on the genotoxic damage produced,

instead of driving the cells towards programmed cell death.


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Diodovich et al. (2004) studied the effect of styrene on cell-cycle gene (c-fos, c-jun)

expression profiles in human cord blood cells and styrene’s effect on production of

apoptosis-related proteins (Bax, Bcl-2, Raf-1). Exposure to styrene at 800 μM for 24 or

48 hours increased necrosis of mononuclear cord blood cells, but not apoptosis. Western

blot analysis revealed induction of both c-jun and c-fos protein, but at different times, as

c-jun was induced early and decreased later, while c-fos was induced only after 48 hours

of exposure to styrene. Production of both Bcl-2 and Raf-1 proteins was induced by

styrene exposure at all time points (6, 24, and 48 hours), whereas Bax protein was

initially downregulated but recovered at the later times. The p53 protein was not

produced in either unexposed or styrene-exposed cells. Macroarray analysis (see

Glossary) showed that styrene-modified cord blood gene expression was associated with

upregulation of monocyte chemotactic protein and downregulation of CC chemokine

receptor type 1 and SLP-76 tyrosine phosphoprotein. The authors concluded that their

results supported a role for styrene in promotion of cell proliferation and cell-cycle

progression, which could potentially favor alterations in gene expression and genotoxic

effects.

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5.5.3 Oxidative stress 17 Marczynski et al. (2000) proposed a mechanism involving oxidative stress and oxidative

DNA damage as the basis for the genotoxic effects of styrene resulting from an

imbalance between oxidants and antioxidants in cells. Gamer et al. (2004) (see Section

5.2.3.2) found no evidence of oxidative stress as indicated by unchanged concentrations

of 8-OH-deoxyguanosine in lung lavage fluid after 20 daily (6 hours per day during a 4-

week period) exposures to styrene at 20, 40, 80, or 160 ppm. However, Roder-Stolinski et

al. (2008) reported that exposure of human lung epithelial cells (cell line A549) to

styrene in vitro stimulated the expression of inflammatory mediators, including

chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) through activation of

the NF-κB signaling pathway, and suggested that activation of the NF-κB signaling

pathway was mediated via a redox-sensitive mechanism (see Section 5.2.1). Cohen et al.

(2002) in their review suggested that the pulmonary hyperplasia that occurs in mice but

not in rats likely results from oxidative damage that is caused either directly by styrene

oxide or indirectly because of depletion of glutathione (GSH). As mentioned above


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(Section 5.5.1) adducts associated with oxidative damage have been reported in styrene-

exposed workers

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5.5.4 Cytotoxic effects of styrene on mouse lung 3 This section discusses cytotoxicity of styrene metabolites as a possible mechanism of

styrene-induced carcinogenesis. Cytotoxicity and cellular proliferation (see Section

5.2.2.2) have been observed, especially in mouse lung Clara cells, following in vitro and

in vivo exposure to styrene and styrene metabolites (styrene-7,8-oxide and 4-

vinylphenol). Chronic cytotoxicity can result in clonal expansion of styrene-induced, or

spontaneous, mutants. Induction of lung tumors resulting from formation of cytotoxic

metabolites has been proposed for other chemicals, including naphthalene. A scientific

panel at the Naphthalene State-of-the-Science Symposium on the pathogenesis of

respiratory tumor formation in rodents (Bogen et al. 2008) hypothesized that nasal

tumors in rats and lung adenomas in mice occur through a cytotoxic mechanism.

Metabolic activation (via Cyp2f2) was required and mouse Clara cells had the greatest

capacity to metabolize naphthalene and were also highly susceptible to naphthalene-

induced cytotoxicity.

Cohen et al. (2002), Cruzan et al. (2002), and IARC (2002) proposed that styrene

exposure causes pulmonary hyperplasia in the mouse lung, which may play a role in the

development of lung tumors. Effects of repeated styrene exposure reported in the lungs of

mice, but not in rats, included focal crowding of bronchiolar cells, bronchiolar epithelial

hyperplasia, and bronchiolo-alveolar hyperplasia (IARC 2002) (see Section 5.2.2.2 for a

description of pneumotoxicity in rodents). Studies by Gadberry et al. (see Section

5.2.2.3) showed that styrene-7,8-oxide administered by i.p. injection caused pulmonary

toxicity in mice, suggesting that styrene-7,8-oxide is responsible for the pneumotoxicity

and that systemically available styrene-7,8-oxide can enter the lung cell. Cohen et al.

postulated that styrene might induce cytotoxicity by directly damaging the cell or by

causing glutathione depletion (see Section 5.2.2.4). Tissue damage leads to hyperplasia,

which makes the tissue more sensitive to tumor development. Cohen et al. (2002) also

stated that the role of hyperplasia does not rule out the possibility that styrene-7,8-oxide

also causes genotoxic effects.


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Cohen et al. (2002) identified three factors that they considered as possible mechanisms

contributing to the development of hyperplasia in mice and subsequent development of

lung tumors: (1) the presence of the cytochromes CYP2E1 and Cyp2f2, which convert

styrene to styrene-7,8-oxide, in mouse lung tissues, (2) greater formation of the R-

enantiomer of styrene-7,8-oxide, which the authors considered the more toxic and

mutagenic of the two enantiomers, and (3) the susceptibility of mouse lung tissue to GSH

depletion, which could reduce the detoxification of styrene-7,8-oxide. After carefully

comparing these factors in rats, mice, and humans (see Section 5.3 for a review of the

literature on interspecies differences, including Cohen et al. 2002), the authors concluded

that these measures of metabolic activity and styrene-7,8-oxide accumulation in the lung

do not explain why styrene caused lung tumors in mice and not rats. IARC (2002)

reached a similar conclusion regarding lung-tumor susceptibility and toxicokinetic

differences between mice and rats.

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Cohen et al. (2002) proposed several reasons why styrene-7,8-oxide production in the

lung did not explain the differences between mice and rats in development of pulmonary

hyperplasia: (1) the Harvard PBPK model did not consistently predict a higher

concentration of styrene-7,8-oxide in the lungs of mice than in rats, (2) styrene-7,8-oxide

concentrations were lower in the blood of mice than in rats by an order of magnitude, and

(3) the predicted concentrations of styrene-7,8-oxide in the lungs were very similar for

mice exposed to styrene by inhalation at 40 to 80 ppm, which resulted in lung tumors,

and rats exposed at 1,000 ppm, which did not induce lung tumors. Cohen et al. also stated

that it was not clear that the pulmonary toxicity of the R-styrene-7,8-oxide was

substantially greater than that of the S-styrene-7,8-oxide; differences in toxicity appear to

be greater in the liver than in the lung (Gadberry et al. 1996, see Section 5.2.4). The

authors also did not think that the greater sensitivity of mice to GSH depletion (see

Section 5.2.2.4) could explain the differences in lung tumor susceptibility, because

styrene caused hyperplasia in mice at concentrations (20 ppm) that do not cause GSH

depletion. Cohen et al. (2002) proposed the following three possible explanations for the

difference in susceptibility: (1) a greater number of Clara cells in mouse pulmonary tissue

than in rat pulmonary tissue, (2) a pharmacokinetic difference at the cellular level, and (3)


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a pharmacodynamic difference, such as greater susceptibility at the cellular level to injury

due to less efficient DNA repair.

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Similar to the first of the three factors put forward by Cohen et al. (2002) as possible

mechanisms for the development of hyperplasia and lung tumors in mice (see above),

Cruzan et al. (2002) proposed that interspecies differences in styrene toxicity are most

likely explained through CYP2F-generated metabolites (2f2 in mice, 2F4 in rats, and 2F1

in humans). They noted that almost all of the effects of cytotoxicity and tumor formation

were seen in tissues that are high in CYP2F isoforms and that CYP2F inhibitors

prevented cytotoxicity (see Section 5.1.3.5). Metabolites formed from ring oxidation,

including 4-vinylphenol, are about 6-fold higher in mice compared with rats, and 4-

vinylphenol has been reported to be more potent than styrene-7,8-oxide as a

pneumotoxicant (see Section 5.1.3.5). Also, styrene metabolism occurs primarily in Clara

cells (see Sections 5.1.3.3 and 5.1.3.5), and mice produce higher levels of toxic

metabolites (R-styrene-7,8-oxide, 4-vinylphenol, and oxidized reactive intermediates of

4-vinylphenol), and have a lower level of detoxifying epoxide hydrolase activity than rats

or humans (see Sections 5.1.3.1 and 5.1.3.2). They stated that PBPK models predicted

that humans do not generate sufficient levels of these metabolites in the terminal

bronchioles to reach toxic levels. Cruzan et al. stated that the tumor profile of styrene

suggests a non-genotoxic mode of action since they felt that the tumors in animals were

common, reported in only one species and one site, did not occur at the 12-month

sacrifice, and were associated with organ toxicity and cell turnover. Studies published

after Cruzan et al.’s 2002 proposal that evaluated the role of Cyp2f2, ring-oxidized

metabolites, and cytotoxicity in the lung are discussed in Sections 5.1.3.5 and 5.2.2.2. For

example, Kaufmann et al. (2005) concluded that the side-chain hydroxylation pathway

appeared to be of minor relevance for the pneumotoxic effects of styrene (see Section

5.2.2.2).

5.5.5 Selected styrene analogues 27 Studies on styrene analogues such ethylbenzene, 1-phenylethanol, 4-methylstyrene, and

vinyltoluene (a mixture of 3- and 4-methylstyrene) (see Table 1-4 for structures of these

analogues), provide further information on the possible relationship between formation of


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ring-oxidized metabolites and the development of lung tumors in experimental animals

exposed to these molecules. [However, no comprehensive reviews or evaluations of all

analogues were identified in the peer-reviewed literature, and thus, only a few analogues

are discussed. No long-term carcinogenicity studies (such as studies in Cyp2f2-knockout

mice) that evaluated this proposed mechanism were identified.]

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Ethylbenzene is a synthetic precursor for styrene (see Section 2.2) differing from styrene

only in the absence of the double bond in the 2-carbon side chain, and 1-phenylethanol is

a metabolite of both ethylbenzene and styrene (see Figure 5-1). Midorikawa et al.

reported that ethylbenzene was metabolized to 1-phenylethanol, 2-ethylphenol, and 4-

methylphenol by rat liver microsomes. The latter two metabolites were metabolically

transformed to the ring-dihydroxylated metabolites ethylhydroquinone and 4-

ethylcatechol (in a separate reaction), respectively, and Midorikawa et al. proposed

further metabolism of the ethylcatechol to 4-ethyl-1,2-benzoquinone. [No in vivo

metabolism studies were identified.] Incubation of 4-ethylcatechol with calf thymus DNA

in vitro resulted in oxidative DNA damage, including the formation of 8-oxo-2'-

deoxyguanosine (8-oxodG) in the presence of Cu(II), and the oxidative stress resulting

from the formation of reactive oxygen species as a result of this proposed metabolic

pathway for ethylbenzene could contribute to the carcinogenic mechanism of

ethylbenzene. (Oxidative stress has been proposed to play a role in styrene-induced

carcinogenicity — see above.) Ethylbenzene has been reported to induce lung tumors in

male mice, liver tumors in female mice, kidney tumors in rats (both sexes), and testicular

tumors in rats (Chan et al. 1998). Stott et al. (2003) reported that chronic exposure to

ethylbenzene induced changes in the mouse lung, including multifocal

bronchiolar/parabronchiolar hyperplasias and altered tinctorial properties. The authors

proposed a nongenotoxic mode of action that was dependent upon cell proliferation and

altered cell population dynamics. However, no studies were identified that evaluated the

role of the ring-oxidized metabolites in lung tumor formation.

Other styrene analogues listed above, i.e., 4-methylstyrene and vinyltoluene, are not

predicted to form 4-phenol metabolites because of the placement of the methyl group at

the 3- or 4- position in these molecules, and no evidence for induction of mouse lung


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tumors by either of these molecules was identified. However, the findings for lung

cytotoxicity were mixed. 4-Methyl styrene (para-methylstyrene) was administered by

gavage to Sprague-Dawley rats (0, 10, 50, 250, and 500 mg/kg per day) and Swiss mice

(0, 10, 50, and 250 mg/kg per day) in a long-term (104 weeks) carcinogenicity study, and

no increased tumor incidence was reported compared with control animals. However,

Conti et al. (1988) reported data only for tumor incidences and did not report any

endpoints for possible lung toxicity. [No inhalation studies were identified.] Vinyltoluene

(a mixture of 65% to 71% 3-[meta-] isomer and 32% to 35% 4-[para-] isomer) was tested

in a two-year inhalation study in F344 rats (0, 100, or 300 ppm) and B6C3F1 mice (0, 10,

or 25 ppm), and no increase in tumor incidences were reported (NTP 1990a). However,

vinyltoluene did cause focal chronic active inflammation and diffuse hyperplasia of the

respiratory epithelium, and chronic active inflammation of the bronchioles.

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No ring-oxidized metabolites of 1-phenylethanol resulting from metabolism of

ethylbenzene by mouse micorsomes were identified in the study by Midorikawa et al.

(2004), and neither lung tumors nor lung cytotoxicity were observed when α-

methylbenzyl alcohol (1-phenylethanol) was administered by gavage to F344 rats and

B6C3F1 mice at doses of 0, 375, or 750 mg/kg per day in a two-year bioassay (NTP

1990b). However, there was an increased incidence of renal tubular-cell adenoma or

adenocarcinoma (combined) in male rats and transitional-cell papillomas of the urinary

bladder occurred in two high-dose female rats. [No inhalation studies with 1-

phenylethanol were identified.]

α-Methylstyrene, another chemical tested in a two-year inhalation study, did not

significantly increase the incidence of lung tumors or cause lung cytotoxicity in mice (in

the two-year study) although it did cause renal tumors and possibly leukemia in male rats

and liver tumors in male (marginal) and female mice (NTP 2007). No metabolism studies

evaluating whether a 4-phenol derivative of this molecule is formed during metabolism

were identified, but its chemical structure does not appear to make that impossible.


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5.6 Summary 1

5.6.1 Absorption, distribution, metabolism, and excretion 2 Styrene can be absorbed through inhalation, ingestion, or skin contact, but the most

important route of exposure in humans in occupational settings is by inhalation, which

results in rapid absorption and distribution of approximately 60% to 70% of inhaled

styrene; the highest tissue concentrations are in subcutaneous fat. Food is also an

important source of exposure for the general population. Metabolic activation of styrene

results in formation primarily of the genotoxic metabolite styrene-7,8-oxide, which can

be detoxified by glutathione conjugation or conversion to styrene glycol by microsomal

epoxide hydrolase. Styrene is metabolized in both the liver and the lung, and the Clara

cells in the lung are regarded as the major cell type in styrene activation following

inhalation exposure. The initial step in styrene metabolism is catalyzed by cytochromes

P450; CYP2E1 and Cyp2f2 are the predominant enzymes in humans and experimental

animals. In animals, CYP2E1 predominates in liver, while Cyp2f2 is the primary enzyme

in mouse lung. CYP2A13, CYP2F1, CYP2S1, CYP3A5, and CYP4B1 are preferentially

expressed in the lung compared with liver in humans, and the human CYP2F1 has been

shown to be capable of metabolizing styrene when expressed in vitro. Because styrene-

7,8-oxide contains a chiral carbon, this and some subsequent styrene metabolites can

exist as either R- or S-enantiomers. A second metabolic pathway through styrene-3,4-

oxide results in formation of 4-vinylphenol, which has been detected in humans, rats, and

mice in vivo, but the importance of 4-vinylphenol in styrene toxicity has not been well

characterized. Almost all absorbed styrene is excreted as urinary metabolites, primarily

mandelic acid and phenylglyoxylic acid.

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5.6.2 Toxicity 24 Styrene exposure has been associated with numerous health effects in humans and

laboratory animals. The acute toxicity of styrene is low to moderate with an oral LD50 of

320 mg/kg and an inhalation LC50 of 4,940 ppm (4-hour exposure) in mice and an oral

LD50 of 5,000 mg/kg and an inhalation LC50 of 2,770 ppm (2-hour exposure) in rats. The

primary effects of acute exposure to styrene in experimental animals and humans include

irritation of the skin, eyes, and respiratory tract and CNS effects. Drowsiness, listlessness,

muscular weakness, and unsteadiness are common signs of systemic styrene intoxication.


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Several studies have reported effects on color vision, hearing threshold, reaction time,

and postural stability following long-term occupational exposure to styrene at

concentrations ranging from about 20 to 30 ppm. Reports of ischemic heart disease and

hepatic, renal, hematological, and immunological effects have been inconsistent. Human

data are insufficient to determine whether styrene is a reproductive or developmental

toxicant, but effects of styrene to increase serum prolactin levels in humans have been

reported.

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Styrene toxicity in experimental animals is similar to that reported in humans. Exposure

to styrene vapors can cause eye and respiratory tract irritation, CNS depression, and

death. Clara cells are the main target of styrene pneumotoxicity, and the available data

indicate increased susceptibility in the mouse. Glutathione depletion as a result of styrene

exposures has been reported to be associated with damage to lung, liver, and kidney

tissues. The cytotoxicity of styrene in the mouse lung, a tissue high in CYP2F isoforms,

could be prevented by CYP2F inhibitors. Some studies have reported reproductive and

developmental effects, but these effects were seen mostly at doses associated with

maternal toxicity. Reported effects have included embryonic, fetal, and neonatal death,

skeletal and kidney abnormalities, decreased birth weight, neurobehavioral abnormalities,

and postnatal developmental delays. The possibility of polystyrene dimer and trimer

extracts from food containers mimicking the physiological effects of estrogen have also

been investigated, but with a mixture of positive and negative results.

5.6.3 Interspecies differences in metabolism, toxicity, and toxicokinetics 21 Species differences exist among rats, mice, and humans in the metabolism and toxicity of

styrene, which may be related, at least in part, to interspecies differences in the

stereochemistry of metabolism. The R-enantiomer, which has been suggested by some

reports to be more toxic than the S-form, has been reported to be produced in relatively

larger amounts in mouse lung than in rat lung, but the difference was less pronounced

when microsomal preparations were used. In mice, the R-isomer of styrene-7,8-oxide was

significantly more hepatotoxic than the S-isomer; the toxicity of the R-isomer also was

greater in the lung, but the difference was not statistically significant.


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5.6.4 Genetic and related effects 1 In vitro studies show that styrene-7,8-oxide forms DNA adducts and causes single-strand

breaks in a dose-related manner. Several studies have shown a correlation between

single-strand breaks and DNA adducts and indicate that the strand breaks, which are not

generally regarded as significantly lethal or mutagenic lesions, are efficiently repaired

within several hours after exposure has stopped. Adducts are formed primarily at the N7-,

N2-, and O6-positions of guanine. N7-adducts are formed in the greatest amount but are

the least persistent, while O6-adducts are formed in the least amount but are the most

persistent. Styrene-7,8-oxide was mutagenic without metabolic activation in all in vitro

mutagenicity test systems reported and caused mutations in some studies in the presence

of metabolizing enzymes. Both styrene and styrene-7,8-oxide caused cytogenetic effects

(sister chromatid exchange [SCE], chromosomal aberrations, and micronuclei) in human

lymphocytes or other mammalian cells in vitro. DNA adducts have been detected in liver

and lung cells of mice and rats exposed to styrene in vivo, although the levels varied

across studies. The majority of studies in experimental animals demonstrated an effect of

both styrene-7,8-oxide and styrene exposure on single-strand breaks, while both positive

and negative results for cytogenetic or clastogenic effects of styrene were reported.

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DNA adducts, primarily N7- and O6-adducts, were reported in white blood cells in all

studies of styrene-exposed workers employed mainly in hand-lamination plants. In most

studies in workers, single-strand breaks showed exposure-related increases; however, two

studies gave negative results. The limited data on mutation frequencies in HPRT and

GPA in styrene-exposed workers are inconclusive. More than half the studies measuring

chromosomal aberrations have reported an increase in chromosomal aberrations in

styrene-exposed workers (or subgroups of workers), and several studies have reported a

positive exposure-response relationship with styrene air levels or urinary metabolites. A

meta-analysis of 22 studies found a positive association between styrene exposure level


greater than or less than a threshold value of 30 ppm for an 8-hour time-weighted

average. Studies of other cytogenetic markers in humans are conflicting. About half of

the studies that evaluated micronucleus and SCE frequency in styrene workers were

positive, and a few studies have reported significant dose-response relationships with


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styrene exposure. A meta-analysis of 10 micronucleus studies was inconclusive, and a

meta-analysis of 14 SCE studies indicated a slight increase in SCE frequency but, again,

was too small to be conclusive. A number of studies have been published on the possible

modulating role of genetic polymorphisms, mainly in xenobiotic metabolism enzymes

and DNA-repair genes, at the level of various biomarkers. Some authors have suggested

that genetic susceptibility (probably at many loci) may be important in styrene-mediated

genotoxicity.

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5.6.5 Mechanistic studies and considerations 8 The proposed mechanisms for the carcinogenicity of styrene include both genotoxic and

epigenetic pathways. These mechanisms, which are not necessarily mutually exclusive,

include: (1) metabolic conversion of styrene to styrene-7,8-oxide and subsequent

induction of DNA damage in the target tissue and (2) cytotoxic effects of styrene

metabolites in the mouse lung. A variety of DNA adducts (including some at base-pairing

sites on nucleotides) induced by styrene and styrene-7,8-oxide has been identified in

human cells, experimental animals, and occupationally exposed workers, but the covalent

binding indices for both molecules are relatively low in rats and mice. The DNA damage

induced by styrene exposure, including single-strand breaks, was found to correlate

significantly with markers of styrene exposure in some studies of styrene workers.

Styrene is mutagenic through the formation of styrene-7,8-oxide (in vitro). A number of

studies reported a positive association between occupational exposure to styrene and the

frequency of chromosomal aberrations, with some studies reporting exposure-response

relationships. Some authors have suggested that polymorphisms in DNA-repair genes

could put some individuals at higher risk for styrene genotoxicity or carcinogenicity.

Many researchers have tried to explain why lung tumors were observed in mice but not in

rats in long-term inhalation exposure studies. Some researchers have proposed that

styrene exposure causes pulmonary hyperplasia in the mouse lung, which may play a role

in the development of lung tumors. Effects of repeated styrene exposure observed in the

lungs of mice, but not in rats, included focal crowding of bronchiolar cells, bronchiolar

epithelial hyperplasia, and bronchiolo-alveolar hyperplasia. The Harvard Center for Risk

Analysis (Cohen et al. 2002) considered three factors as possible explanations for the


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greater susceptibility of mouse lung than rat lung to development of hyperplasia leading

to tumors with exposure to styrene are: (1) the presence of the styrene-metabolizing

cytochromes in mouse lung tissues, (2) greater formation of the R-enantiomer of styrene-

7,8-oxide, and (3) the susceptibility of mouse lung tissue to glutathione depletion.

However, they concluded that although toxicokinetic models generally predict higher

rates of metabolism by mice and rats than by humans, the models do not consistently

predict a difference between the rodent species. An alternative mechanism is that

interspecies differences in styrene toxicity are most likely explained through CYP2F-

generated metabolites (2f2 in mice, 2F4 in rats, and 2F1 in humans) in the mouse lung.

This is based on data showing that most of the effects of cytotoxicity and tumor

formation were seen in mouse respiratory tissues, which are high in CYP2F isoforms, and

that CYP2F inhibitors prevented cytotoxicity. Moreover, metabolites formed from ring

oxidation, including 4-vinylphenol, are about 6-fold higher in mice compared with rats,

and 4-vinylphenol is more potent than styrene-7,8-oxide as a pneumotoxicant.

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6 References

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515. Vodicka P, Vodicková L, Trejbalová K, Srám RJ, Hemminki K. 1994. Persistence of O6-guanine DNA adducts in styrene-exposed lamination workers determined by 32P-postlabelling. Carcinogenesis 15(9): 1949-1953. (Supported by the EC Environment Program, the Swedish Medical Research Council, the National Environmental Protection Board and the Swedish Cancer Fund. Authors affiliated with Czech Academy of Sciences, Czech Republic; Regional Hygienic Station, Czech Republic; National Institute of Public Health, Czech Republic; Karolinska Institute, Sweden.)

516. Vodicka P, Bastlová T, Vodicková L, Peterková K, Lambert B, Hemminki K. 1995. Biomarkers of styrene exposure in lamination workers: levels of O6-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes. Carcinogenesis 16(7): 1473-1481. (Supported by the Swedish Environmental Protection Board, the Swedish Cancer Society, the Swedish Work Environmental Fund and the EU Environment Program. Authors affiliated with Czech Academy of Sciences, Czech Republic; Regional Institute of Hygiene, Czech Republic; Karolinska Institute, Sweden; National Institute of Public Health, Czech Republic.)

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517. Vodicka P, Stetina R, Kumar R, Plna K, Hemminki K. 1996. 7-Alkylguanine adducts of styrene oxide determined by 32P-postlabeling in DNA and human embryonal lung fibroblasts (HEL). Carcinogenesis 17(4): 801-808. (Supported by the EU Environment, PECO Program, Swedish Medical Council, National Environmental Protection Board, Swedish Cancer Fund and the Czech Ministry of Health. Authors affiliated with Czech Academy of Science, Czech Republic; Karolinska Institute, Sweden.)

518. Vodicka P, Tvrdik T, Osterman-Golkar S, Vodicková L, Peterková K, Soucek P, Šarmanová J, Farmer PB, Granath F, Lambert B, Hemminki K. 1999. An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers. Mutat Res 445(2): 205-224. (Supported by the EU Environment, PECO Program, Swedish Medical Council, Swedish National Environment Protection Board, Swedish Cancer Society, and the Czech Ministry of Health. Authors affiliated with Czech Academy of Sciences, Czech Republic; Karolinska Institute, Sweden; Stockholm University, Sweden; National Institute of Public Health, Czech Republic; University of Leicester, UK.)

519. Vodicka P, Soucek P, Tates AD, Dusinska M, Sarmanova J, Zamecnikova M, Vodickova L, Koskinen M, de Zwart FA, Natarajan AT, Hemminki K. 2001a. Association between genetic polymorphisms and biomarkers in styrene-exposed workers. Mutat Res 482(1-2): 89-103. (Supported by EU, GACR and the Swedish Council for Work Life Research. Authors affiliated with Czech Academy of Sciences, Czech Republic; National Institute of Public Health, Czech Republic; Leiden University Medical Center, Netherlands; Institute of Preventive and Clinical Medicine, Slovak Republic; National Institute of Public Health, Slovak Republic; Karolinska Institute, Sweden.)

520. Vodicka P, Koskinen M, Vodicková L, Štetina R, Šmerák P, Bárta I, Hemminki K. 2001b. DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation. Chem Biol Interact 137(3): 213-227. (Supported by the European Communites, GACR and the Swedish Council for Work Life Research. Authors affiliated with Academy of Sciences of the Czech Republic; Karolinska Institute, Sweden; National Institute of Public Health, Czech Republic; Purkynje Military Medical Academy, Czech Republic; Charles University, Czech Republic.)

521. Vodicka P, Koskinen M, Arand M, Oesch F, Hemminki K. 2002a. Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers and prospects in human biomonitoring. Mutat Res 511(3): 239-254. (Supported by the European Communities, GACR and the Swedish Council for Work Life Research. Authors affiliated with Academic Sciences of the Czech Republic; Karolinska Institute, Sweden; University of Mainz, Germany; Orion Pharma, Finland.)

522. Vodicka P, Stetina R, Koskinen M, Soucek P, Vodickova L, Hlavac P, Kuricova M, Necasova R, Hemminki K. 2002b. New aspects in the biomonitoring of occupational exposure to styrene. Int Arch Occup Environ Health 75(85): S75-85. (Supported by the Academy of Sciences of the Czech Republic, Grant Agency of

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the Czech Republic and the European Union. Authors affiliated with Academy of Sciences of the Czech Republic; Purkyne Military Medical Academy, Czech Republic; Orion Pharma, Finland; National Institute of Public Health, Czech Republic; Regional Hygiene Station, Czech Republic; Institute of Preventive and Clinical Medicine, Slovak Republic; Karolinska Institute, Sweden.)

523. Vodicka P, Koskinen M, Stetina R, Soucek P, Vodickova L, Matousu Z, Kuricova M, Hemminki K. 2003. The role of various biomarkers in the evaluation of styrene genotoxicity. Cancer Detect Prev 27(4): 275-284. (Supported by the Academy of Sciences of the Czech Republic, Grant Agency of the Czech Republic, and the European Union. Authors affiliated with Academy of Science of the Czech Republic; Purkynje Military Medical Academy, Czech Republic; National Institute of Public Health, Czech Republic; Institute of Preventive and Clinical Medicine, Slovak Republic; Orion Pharma, Finland; Karolinska Institutem Sweden.)

524. Vodicka P, Tuimala J, Stetina R, Kumar R, Manini P, Naccarati A, Maestri L, Vodickova L, Kuricova M, Jarventaus H, Majvaldova Z, Hirvonen A, Imbriani M, Mutti A, Migliore L, Norppa H, Hemminki K. 2004a. Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers. Environ Health Perspect 112(8): 867-871. (Supported by the Academy of Sciences of the Czech Republic, Grant Agency of the Czech Republic and the European Union. Authors affiliated with Academy of Science of the Czech Republic; Finnish Institute of Occupational Health, Finland; Purknyje Military Medical Academy, Czech Republic; Karolinska Institute, Sweden; German Cancer Institute, Germany; University of Parma, Italy; University of Pisa, Italy; University of Pavia, Italy; National Institute of Public Health, Czech Republic; Regional Hygiene Station, Czech Republic.)

525. Vodicka P, Kumar R, Stetina R, Musak L, Soucek P, Haufroid V, Sasiadek M, Vodickova L, Naccarati A, Sedikova J, Sanyal S, Kuricova M, Brsiak V, Norppa H, Buchancova J, Hemminki K. 2004c. Markers of individual susceptibility and DNA repair rate in workers exposed to xenobiotics in a tire plant. Environ Mol Mutagen 44: 283-292. (Supported by the Grant Agency of the Czech Republic and the European Center for Ecotoxicology and Toxicology of Chemicals. Authors affiliated with Academy of Science of the Czech Republic, Czech Republic; German Cancer Research Center, Germany; Purkynje Military Medical Academy, Czech Republic; Jessenius Medical Faculty, Slovak Republic; National Institute of Public Health, Czech Republic; Universite Cathloique de Louvain, Belgium; Wroclaw Medical University, Poland; Regional Hygenic Station, Slovak Republic; Finnish Institute of Occupational Health, Finland.)

526. Vodicka P, Koskinen M, Naccarati A, Oesch-Bartlomowicz B, Vodickova L, Hemminki K, Oesch F. 2006b. Styrene metabolism, genotoxicity, and potential carcinogenicity. Drug Metab Rev 38(4): 805-53. (Supported by AVOZ and GACR. Authors affiliated with Academy of Sciences of the Czech Republic, Czech Republic; Orion Pharma, Finland; Johannes Gutenberg University, Germany;

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German Cancer Research Center, Germany; National Institute of Public Health, Czech Republic; Karolinska Institute, Sweden.)

527. Vodicka PE, Linhart I, Novak J, Koskinen M, Vodickova L, Hemminki K. 2006a. 7-Alkylguanine adduct levels in urine, lungs and liver of mice exposed to styrene by inhalation. Toxicol Appl Pharmacol 210(1-2): 1-8. (Supported by GACR and AVOZ. Authors affiliated with Academy of Sciences of Czech Republic, Czech Republic; Institute of Chemical Technology Prague, Czech Republic; Orion Pharma, Finland; National Institute of Public Health, Czech Republic; German Cancer Research Center, Germany; Karolinska Institute, Sweden.)

528. Vogie K, Mantick N, Carlson G. 2004. Metabolism and toxicity of the styrene metabolite 4-vinylphenol in CYP2E1 knockout mice. J Toxicol Environ Health A 67(2): 145-52. (Supported by the Styrene Information and Research Center. Authors affiliated with Purdue Unversity, IN.)

529. von der Hude W, Carstensen S, Obe G. 1991. Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells. Mutat Res 249(1): 55-70. (Supported by the Umweltbundesamt (Federal Environmental Agency.) Authors affiliated with Freie Universität, Germany; Universität-GH Essen, Germany.)

530. Walles SA, Edling C, Anundi H, Johanson G. 1993. Exposure dependent increase in DNA single strand breaks in leucocytes from workers exposed to low concentrations of styrene. Br J Ind Med 50(6): 570-4. (Support not reported. Authors affiliated with National Institute of Occupational Health, Sweden; University Hospital, Sweden.)

531. Walles SAS, Orsen I. 1983. Single-strand breaks in DNA of various organs of mice induced by styrene and styrene oxide. Cancer Lett 21: 9-15. (Support not reported. Authors affiliated with National Board of Occupational Safety and Health, Sweden.)

532. Watanabe T, Endo A, Sato K, Ohtsuki T, Miyasaka M, Koizumi A, Ikeda M. 1981. Mutagenic potential of styrene in man. Ind Health 19(1): 37-45. (Support not reported. Authors affiliated with Yamagata University School of Medicine, Japan; Kyoto Industrial Health Association, Japan; Tohoku University School of Medicine, Japan.)

533. Watanabe T, Endo A, Kumai M, Ikeda M. 1983. Chromosome aberrations and sister chromatid exchanges in styrene-exposed workers with reference to their smoking habits. Environ Mutagen 5(3): 299-309. (Supported by the Ministry of Education, Science and Culture of the Government of Japan. Authors affiliated with Yamagata University School of Medicine, Japan; Tohoku University School of Medicine, Japan.)

534. Wenker MA, Kežic S, Monster AC, de Wolff FA. 2000. Metabolism of styrene-7,8-oxide in human liver in vitro: interindividual variation and stereochemistry. Toxicol Appl Pharmacol 169(1): 52-58. (Support not reported. Authors affiliated with

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University of Amsterdam, Netherlands; Leiden University Medical Center, Netherlands.)

535. Wenker MA, Kezic S, Monster AC, de Wolff FA. 2001a. Stereochemical metabolism of styrene in volunteers. Int Arch Occup Environ Health 74(5): 359-65. (Support not reported. Authors affiliated with University of Amsterdam, Netherlands; Ledien University Medical Center, Netherlands.)

536. Wenker MA, Kezic S, Monster AC, De Wolff FA. 2001b. Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity. Xenobiotica 31(2): 61-72. (Support not reported. Authors affiliated with Coronel Institute, Netherlands; Leiden University Medical Center, Netherlands; University of Amsterdam, Netherlands.)

537. Wenker MA, Kezic S, Monster AC, de Wolff FA. 2001c. Metabolic capacity and interindividual variation in toxicokinetics of styrene in volunteers. Hum Exp Toxicol 20(5): 221-8. (Support not reported. Authors affiliated with University of Amsterdam, Netherlands; Leiden University Medical Center, Netherlands; NOTOX Safety and Environmental Research, Netherlands.)

538. WHO. 1983. Styrene. Environmental Health Criteria: 26. Geneva: World Health Organization. 123 pp. http://www.inchem.org/documents/ehc/ehc/ehc26.htm.

539. Wieczorek H. 1985. Evaluation of low exposure to styrene. II. Dermal absorption of styrene vapours in humans under experimental conditions. Int Arch Occup Environ Health 57(1): 71-75. (Support not reported. Authors affiliated with Institute of Occupational Medicine in the Textile and Chemical Industries, Poland.)

540. Wieczorek H, Piotrowski JK. 1988. Kinetic interpretation of the exposure test for styrene. Int Arch Occup Environ Health 61(1-2): 107-113. (Support not reported. Authors affiliated with Nofer's Institute of Occupational Medicine, Poland; Medical Academy of Łódź, Poland.)

541. Wolf MA, Rowe VK, McCollister DD, Hollingsworth RL, Oyen F. 1956. Toxicological studies of certain alkylated benzenes and benzene: experiments on laboratory animals. AMA Arch Ind Health 14: 387-398. (Support not reported. Authors affiliated with Dow Chemical Company.)

542. Wong O. 1990. A cohort mortality study and a case-control study of workers potentially exposed to styrene in the reinforced plastics and composites industry. Br J Ind Med 47(11): 753-762. (Support not reported. Authors affiliated with ENSR Health Sciences, CA.)

543. Wong O, Trent LS, Whorton MD. 1994. An updated cohort mortality study of workers exposed to styrene in the reinforced plastics and composites industry. Occup Environ Med 51(6): 386-396. (Supported by the Styrene Information and Research Center. Authors affiliated with Applied Health Sciences, CA; ENSR, CA.)

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http://www.inchem.org/documents/ehc/ehc/ehc26.htm


544. Yager JW, Paradisin WM, Rappaport SM. 1993. Sister-chromatid exchanges in lymphocytes are increased in relation to longitudinally measured occupational exposure to low concentrations of styrene. Mutat Res 319(3): 155-65. (Supported by NIOSH, CDC and NIEHS. Authors affiliated with University of California Berkely, CA; Electric Power Research Institute, CA; Schering-Plough, NJ; University of North Carolina, NC.)

545. Yeowell-O'Connell K, Jin Z, Rappaport SM. 1996. Determination of albumin and hemoglobin adducts in workers exposed to styrene and styrene oxide. Cancer Epidemiol Biomarkers Prev 5(3): 205-215. (Support not reported. Authors affiliated with University of North Carolina, NC.)

546. Yuan W, Chung J, Gee S, Hammock BD, Zheng J. 2007. Development of polyclonal antibodies for the detection of styrene oxide modified proteins. Chem Res Toxicol 20(2): 316-21. (Supported by NIH and NIEHS. Authors affiliated with Northwestern University, MA; University of California Davis, CA; University of Washington, WA.)

547. Yunis JJ. 1983. The chromosomal basis of human neoplasia. Science 221(4607): 227-36. (Support not reported. Authors affiliated with University of Minnesota, MN.)

548. Zang H, Harris TM, Guengerich FP. 2005b. Kinetics of nucleotide incorporation opposite DNA bulky guanine N2 adducts by processive bacteriophage T7 DNA polymerase (exonuclease-) and HIV-1 reverse transcriptase. J Biol Chem 280(2): 1165-1178. (Supported by the U.S. Public Health Service. Authors affiliated with Vanderbilt University School of Medicine, TN.)

549. Zhang JY, Wang Y, Prakash C. 2006. Xenobiotic-metabolizing enzymes in human lung. Curr Drug Metab 7(8): 939-48. (Support not reported. Authors affiliated with GlaxoSmithKline, PA; Bristol-Meyers Squibb, NJ; Pfizer Global Research and Development, CT.)

550. Zhang W, Johnson F, Grollman AP, Shibutani S. 1995. Miscoding by the exocyclic and related DNA adducts 3,N4-etheno-2'-deoxycytidine, 3,N4-ethano-2'-deoxycytidine, and 3-(2-hydroxyethyl)-2'-deoxyuridine. Chem Res Toxicol 8(1): 157-163. (Supported by NIH. Authors affiliated with State University of New York at Stony Brook, NY.)

551. Zhang XX, Chakrabarti S, Malick AM, Richer CL. 1993. Effects of different styrene metabolites on cytotoxicity, sister-chromatid exchanges and cell-cycle kinetics in human whole blood lymphocytes in vitro. Mutat Res 302(4): 213-8. (Supported by the Institut de Recherche en santé et en sécurité du Travail, Quebec. Authors affiliated with Université de Montréal, Canada.)

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Glossary of Terms Acinar: Pertaining to one of the granular masses which constitute a racemose or

compound gland such as the pancreas.

Acute: The clinical term is used for a disease having a short and relatively severe course. In rodent testing, usually pertains to administration of an agent in a single dose.

Adduct: A complex that forms when a chemical binds to a biological molecule such as DNA or a protein.

Adenocarcinomas: A cancer that develops in the lining or inner surface of an organ.

Adenoma: An ordinarily benign neoplasm of epithelial tissue in which the neoplastic cells form glands or gland-like structures in the stroma.

Adipose tissue: Fatty tissue.

Allele: Any one of a series of two or more different genes that occupy the same position (locus) on a chromosome.

Alveolar/bronchiolar: Pertaining to the alveoli or bronchi of the lungs.

Ambient air: Outdoor air to which the general public is exposed.

Aneuploidy: One or a few chromosomes above or below the normal chromosome number.

Apoptosis: Cell deletion by fragmentation into membrane-bound particles which are phagocytosed by other cells.

Aquifer: Geologic formations containing sufficient saturated porous and permeable material to transmit water.

Aromatic hydrocarbon: An organic chemical compound formed primarily from carbon and hydrogen atoms with a structure based on benzene rings and resembling benzene in chemical behavior; substituents on the rings(s) may contain atoms other than carbon or hydrogen.

Autoignition: The temperature at or above which a material will spontaneously ignite (catch fire) without an external spark or flame.

Benign tumor: An abnormal mass of tissue that does not spread and that is not life-threatening.

Bilirubin: A pigment produced when the liver processes waste products.

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Bioaccumulation: The process by which a material in an organism's environment progressively concentrates within the organism.

Bioassay: The determination of the potency or concentration of a compound by its effect upon animals: Isolated tissues: Or microorganisms: As compared with a chemical or physical assay.

Bioconcentrate: Accumulation of a chemical in tissues of a fish or other organism to levels greater than in the surrounding medium.

Biodegradation: Biotransformation; the conversion within an organism of molecules from one form to another: A change often associated with change in pharmacologic activity.

Bronchioloalveolar: Derived from epithelium of terminal bronchioles.

Carcinoma: A malignant neoplasm of the epithelium.

Chopper gun: A device that feeds fiber glass rovings through a chopper and ejects them into a stream of resin and organic peroxide catalyst onto a mold surface.

Chromosomal aberrations: Any abnormality of a chromosome's number or structure.

Chronic: Continuing for a long period time. In rodent testing, pertains to dosing schedules of greater than 3 months.

Clara cells: Unciliated cells found in the epithelium of the respiratory and terminal bronchioles.

Clastogen: Any substance which causes chromosomal breaks.

Confounding: A relationship between the effects of two or more causal factors observed in a set of data such that it is not logically possible to separate the contribution of any single causal factor to the observed effects.

Copolymers: A polymer of two or more different monomers.

Creatinine: A waste product of protein metabolism that is found in the urine.

Critical temperature: The temperature of a gas above which it is no longer possible by use of any pressure: However great: To convert it into a liquid.

Cytogenetic: The cellular constituents concerned in heredity.

Dam: Female parent.

Dehydrogenation: The removal of one or more hydrogen ions or protons from a molecule.

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Diffusion coefficient: The rate at which a substance moves from an area of high concentration to an area of low concentration.

Dimroth rearrangement : Rearrangement whereby exo- and endocyclic heteroatoms on a heterocyclic ring are translocated.

Dissociation constant (pka): The equilibrium constant for the breaking apart of a weak acid into its hydrogen and conjugate base in a water solution.

Effluents: Waste material such as water from sewage treatment or manufacturing plants discharged into the environment.

Enantiomer: One of a pair of compounds having a mirror image relationship.

Endogenous: Originating within an organism.

Epidemiology: A science concerned with the occurrence and distribution of disease in populations.

Epididymis: The epididymis is a coiled segment of the spermatic ducts that serves to store and transport spermatozoa between the testis and the vas deferens.

Epithelial: Relating to or consisting of epithelium.

Erythema: Redness of the skin produced by congestion of the capillaries.

Erythrocytes: Cells that carry oxygen to all parts of the body (red blood cells).

Eukaryote: An organism whose cells contain a limiting membrane around the nuclear material and which undergoes mitosis.

Ever hourly: Workers who had ever worked in an hourly job.

Explosive limit: The concentration range in which a flammable substance can produce and explosion or fire when an ignition source (such as a spark or open flame) is present. The concentration is usually expressed as percent fuel by volume. Below the lower explosive limit (also called lower flammable limit or LFL) the mixture of the substance and air lacks sufficient fuel to burn, while above the upper explosive limit (upper flammable limit or UFL) the mixture is too rich in fuel (i.e., deficient in oxygen) to burn.

Extrahepatic: Outside of, or unrelated to, the liver.

Fibroblasts: Connective tissue cells.

Flash point: The lowest temperature at which a liquid can form an ignitable mixture in air near the survace of the liquid.

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Gavage: In animal experiments, the introduction of material through a tube passed through the mouth into the stomach.

Genotoxicity: The amount of damage caused to a DNA molecule.

Hematopoietic: Pertaining to the formation of blood or blood cells.

Half-life: The time required for a substance to be reduced to one-half its present value through degradation or through elimination from an organism.

Henry’s law: The relationship that defines the partition of a soluble or partially soluble species between the gas and solution phases.

Hepatoblastoma: A malignant neoplasm occurring in young children, primarily in the liver, composed of tissue resembling embryonal or fetal hepatic epithelium, or mixed epithelial and mesenchymal tissues.

Hepatocellular: Pertaining to cells of the liver.

Heterozygotes: An organism that has different alleles at a particular gene locus on homologous chromosomes.

Hodgkin’s disease: A form of malignant lymphoma characterized by painless progressive enlargement of the lymph nodes, spleen, and general lymphoid tissue.

Homozygotes: An organism that has the same alleles at a particular gene locus on homologous chromosomes.

Hydrolysis: The chemical breakdown of a compound due to reaction with water.

Hydroxyl radicals: A particularly reactive, damaging type of free radical that is formed when superoxide radicals react with hydrogen peroxide.

In vitro: Biological process taking place in a test tube: Culture dish: Or elsewhere outside a living organism.

In vivo: Biological processes taking place in a living organism.

Intraperitoneal [i.p.] injection: Injection within the peritoneal cavity, i.e., the area that contains the abdominal organs.

Isoenzymes: Any of the chemically distinct forms of an enzyme that perform the same biochemical function.

Koc (soil organic carbon-water partitioning coefficient): A measure of the tendency for organics to be adsorbed by soil and sediment which is useful in predicting the mobility of organic contaminants in soil.

LD50: The dose that kills 50 percent of a group of test animals.

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Leachate: The liquid produced in a landfill from the decomposition of waste within the landfill.

Leukemia: A cancer of the blood-forming tissues that is characterized by a marked increase in the number of abnormal white blood cells (leukocytes).

Lipophilicity: The affinity of a molecule or a moiety for a lipophilic (as fats) environment.

Lymphatic: A small sac or node in which lymph is stored; or pertaining to the lymph, lymph nodes, or vascular channels that transport lymph to the lymph nodes.

Lymphohaematopoietic: Of, relating to, or involved in the production of lymphocytes and cells of blood, bone marrow, spleen, lymph nodes, and thymus.

Lymphoma: A neoplasm of the lymphatic tissue.

Lymphosarcoma: Any of various malignant neoplastic disorders of lymphoid tissue; excluding Hodgkin's disease.

Macroarray: A term for microarrays with larger and fewer spots in the array.

Macrophage: A large cell that is present in blood, lymph, and connective tissues, removing waste products, harmful microorganisms, and foreign material from the bloodstream.

Malignant: Tending to become progressively worse; life-threatening.

Metabolism: The whole range of biochemical processes that occur within living organisms, consisting both of anabolism and catabolism (the buildup and breakdown of substances, respectively).

Metabolite: A substance produced by metabolism.

Micronuclei: Nuclei separate from, and additional to, the main nucleus of a cell, produced during the telophase of mitosis or meiosis by lagging chromosomes or chromosome fragments derived from spontaneous or experimentally induced chromosomal structural changes.

Monomer: A chemical subunit that is joined to other similar subunits so as to produce a polymer.

Multiple myeloma: A malignant neoplasm derived from plasma cells and found at several locations in the body.

Necropsy: The examination of the dead body of an animal by dissection so as to detail the effects of the disease.

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Necrosis: The pathologic death of one or more cells, or of a portion of tissue or organ, resulting from irreversible damage.

Neoplasm: An abnormal mass of cells.

Non-Hodgkin’s lymphoma: A heterogeneous group of malignant lymphomas; the only common feature being an absence of the giant Reed-Sternberg cells characteristic of Hodgkin's disease.

Nucleoside: An organic compound consisting of a purine or pyrimidine base linked to a sugar but lacking the phosphate residues that would make it a nucleotide.

Nucleotide: The molecular subunit of nucleic acids; consists of a purine or pyrimidine base, a sugar, and phosphoric acid.

Octanol-water partition coefficient (Kow): A measure of the equilibrium concentration of a compound between octanol and water.

Parenchyma: The distinguishing or specific cells of a gland or organ, contained in and supported by the connective tissue, framework, or stroma.

Percutaneous: Effected or performed through the skin.

Perirenal: Of, relating to, occurring in, or being the tissues surrounding the kidney.

Polymer: A chemical formed by the joining together of similar chemical subunits.

Polymorphism: A variation in the DNA that is too common to be due merely to new mutation.

Pyknosis: Contraction of nuclear contents to a deep staining irregular mass; a sign of cell death.

Racemic: Denoting a mixture that is optically inactive, being composed of an equal number of dextro- and levorotary substances which are separable.

Resin: Any of numerous physically similar polymerized synthetics or chemically modified natural resins including thermoplastic materials such as polyvinyl, polystyrene, and polyethylene and thermosetting materials such as polyesters, epoxies, and silicones that are used with fillers, stabilizers, pigments, and other components to form plastics.

Sister chromatid exchange (SCE): The exchange during mitosis of homologous genetic material between sister chromatids; increased as a result of inordinate chromosomal fragility due to genetic or environmental factors.

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Subacute: Between acute and chronic; denoting the course of a disease of moderate duration or severity. In rodent testing, usually pertains to a dosing schedule of less than one month.

Subchronic: In rodent testing, generally refers to a dosing schedule lasting from one to three months.

Subcutaneous injection: Injection beneath the skin.

Threshold limit value (TLV): The maximum permissible concentration of a material, generally expressed in parts per million in air for some defined period of time.

Time-weighted average (TWA): The average exposure concentration of a chemical measured over a period of time (not an instantaneous concentration).

Vacuolation: Creation of small cavities containing air or fluid in the tissues of an organism.

Vapor density: The ratio of the weight of a given volume of one gas to the weight of an equal volume of another gas at the same temperature and pressure.

Vapor pressure: The pressure exerted by a vapor in equilibrium with its solid or liquid phase.

Volatile: Quality of a solid or liquid allowing it to pass into the vapor state at a given temperature.

Xenobiotic: A pharmacologically, endocrinologically, or toxicologically active substance not endogenously produced and therefore foreign to an organism.

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Erratum and Addendum to the Final Report on Carcinogens Background Document for Styrene

The following corrections are made to the Final Report on Carcinogens Background Document for Styrene.

1. Table 3-10, page 187. The footnote * is corrected to read: “Note that Kolstad et al. classified employees at companies with 50% or more of workers involved in reinforced plastics as probable high exposure, and that most of the companies were boat yards or manufacturers of containers by hand lamination.”

2. Page 174, lines 12-13. The following text in parentheses, “(workers from plants

employing 50% to 100% laminators),” is deleted from the sentence: “Kolstad et al. (1995) reported significant risks of pancreatic cancer among individuals with probable high styrene exposures (workers from plants employing 50% to 100% laminators), and among individuals exposed to styrene for greater than one year.” The following sentence is added: “The authors classified employees at companies with 50% or more of workers involved in reinforced plastics as probable high exposure, and most of the companies were boat yards or manufacturers of containers by hand lamination.”

3. Page 384, lines 7-9. The reference is added: “An alternative mechanism (Cruzan et al. 2002) is that interspecies differences in styrene toxicity are most likely explained through CYP2F-generated metabolites (2f2 in mice, 2F4 in rats, and 2F1 in humans) in the mouse lung.”

The following clarifications are made to the Final Report on Carcinogens Background Document for Styrene. New text is shown in italics.

1. The terms “statistically significant” and/or “statistically non-significant” and/or the P value are added to clarify the reported findings as follows: • Page xii, lines 19-22 and page 192, lines 14-17: “In the styrene monomer and

polymer industries, the risk of lymphohematopoietic malignancies was also increased (both statistically significant and statistically non-significant) in most of the studies (as well as the total number of observed cases across studies), but these workers might also have been exposed to benzene.”

• Page xii, line 30 to page xiii, line 2 and page 192, lines 25 to 27: “The risk of pancreatic cancer was slightly higher among the Danish workers with longer term employment and earlier start date, and increased with cumulative exposure (P = 0.068) in the multi-plant cohort.”

• Page 178, lines 27-30: “In analyses of subtypes of leukemia, the risk of myelogenous leukemia (chronic and acute) was slightly higher than for all leukemia (Kogevinas et al.1994a), and statistically non-significant increased risk was also seen for myeloid leukemia with chromosomal aberrations in a nested case-control study of the Danish workers based on small number of cases (Kolstad et al. 1996).”

2

• Page 181, lines 19-24: “The nested case-control study from the Matanoski cohort of 58 lymphohematopoietic cases and 1,242 controls found two- to three-fold statistically significant increased risks for lymphoma, lymphosarcoma, and myeloma and styrene exposure (increase of 1 ppm in TWA) (Matanoski et al. 1997), and the risk of myeloma increased with increasing cumulative exposure (P = 0.023) to styrene using the step-down regression analysis and taking into account butadiene exposure and other variables.

• Page 184, lines 20-22: “A statistically significant increased risk of renal-cell cancer was also associated with exposure to styrene-butadiene rubber in the population case-control study from Canada (Parent et al. 2000).”

• Page 184, lines 25-29: “Statistically significant increased risk of breast cancer was suggested in an ecological study (Coyle et al. 2005), which assessed styrene exposure by toxic release inventory data; [however, this study was limited by the ecological design and poor characterization of styrene exposure, which was based only on residence in counts with varying environmental toxic releases].”

2. Page 360, lines 17-18. The following sentence is deleted: “However, most of the studies

published prior to 1994 were negative while most of the studies published after 1994 were positive.”

3. Table 5-18, page 367. The designation for Mutations – In vivo Humans is changed from

“(+)” to “inconclusive.”

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